Vitamin Compendium - The Properties of The - Hoffmann-La Roche (F.)

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Vitamin Compendium
The Properties of the Vitamins

and their Importance in Human and Animal Nutrition
ROCHE
Vitamins and Chemicals Department

F. Hoffmann-La Roche & Co. Ltd, Basle, Switzerland
6
Contents
Part I: General
7 The Physiological Importance of the Vitamins for Man and Animals

1 Vitamins in Human Medicine
19 Vitamins and Carotenoids in Food
28 Vitamins and Carotenoids in Animal Nutrition
40 Technological Properties and Formulations of the Vitamins
Part II: The Individual Vitamins
(Physico-chemical properties, methods of determination, biological

units and standardisation, physiological functions, deficiency symp-
toms, requirements)
49 Vitamin A
59 Provitamin A
62 Vitamin D
68 Vitamin E
77 Vitamin K
83 Vitamin C
89 Vitamin Bi
96 Vitamin B2
102 Vitamin 65
no Vitamin Bjj
116 Nicotinic Acid and Nicotinamide
123 Pantothenic Acid
132 Biotin
139 Folic Acid
144 Bibliography
Part I: General
: :
The Physiological Importance

of the Vitamins in Man and Animals
Essential Human and animal organisms require, for the proper operation of all
dietary of their physiological functions, a regular intake of some forty different

components dietary components. If only one of these is missing or its supply is in-
adequate, deficiency symptoms appear which, if prolonged, can be fatal.
All these components of the diet are therefore indispensablefor life (''essential" ) ;
they comprise the actual energy-yielding and body-building substances

(proteins, fats, carbohydrates, amino-acids, mineral salts) and also the
"micro"-nutrients (vitamins, trace elements).
Vitamins The vitamins are therefore active substances essential for life. As a
essential dietary group, they are recognised by two characteristic properties

components
1. The daily requirement for each vitamin for an individual is very
small, usually measured in microgrammes or milligrammes in this respect ;
they differ from the " macro "-nutrients which are required in at least
looo times larger amounts. Vitamins, on the other hand, are mediators of
syntheticand degradative processes without serving as building substances
themselves.
2. Vitamins are compounds, differing in this respect from the
organic
manganese and zinc which, however,
trace elements such as iron, iodine,
are also essential compounds.
To-day, 1 3 vitamins are known, each of which represents a group of
related compounds with the same qualitative activity. The provitamin A
group is also of great practical importance; this comprises compounds
which are partly transformed into vitamin A in the organism. The Table
(p. 8/9) surveys the compounds with vitamin properties.
In addition to these 1 3 vitamins, there are other substances which have
been classed with the vitamins although their vitamin character has not
yet been established. Examples are: orotic acid (vitamin 6,3); inositol or
Bios I; lipoic acid or thioctic acid; rutin (vitamin P); xanthopterin (vit-
amin B14); carnitine (vitamin Bt); pangamic acid (vitamin B,,); and
ubiquinone (coenzyme Q).
Unidentified In this connection, the so-called "unidentified growth factors" (U.G.F.)

growth factors which, under certain conditions, can favourably affect the growth and
productivity of livestock, should be mentioned. They are present, for
example, in residues of alcohol fermentation, in fish-solubles, grass sap,

whey and egg-yolk. Nothing is yet known of the chemical nature of these
unidentified growth factors. They may perhaps not be single entities but
mixtures of known essential nutritional factors with mutually potentiating
effects (synergism).
; ; ; ;
The physiological importance of the vitamins for man and animals
Vitamins: Active Substances and Commercial Forms
Vitamin Group Principal Principal

Representatives Commercial Forms
Vitamin A Vitamin A (-alcohol) Vitamin A-acetate

(Anti-xerophthalmic (Retinol; Axerophthol) Vitamin A-palmitate
vitamin; anti-infective Vitamin A, -aldehyde
vitamin; skin- (Retinal; retinene)
protective vitamin; Vitamin A, (-alcohol)
growth vitamin) (
3 -Dehy droretinol)
Provitamin A /3-Carotene /5-Carotene

y-Carotene
Vitamin D Vitamin D^ Vitamin D2

(anti-rachitic (Ergocalciferol) Vitamin D,
vitamin) Vitamin D,
(Cholecalciferol)
Vitamin E (anti- a-Tocopherol d-a-Tocopherol

dystrophic vitamin; /3-Tocopherol dl-a-Tocopherol
anti-sterility vitamin d-a-Tocopheryl acetate
fertility vitamin) dl-a-Tocopheryl acetate
Vitamin K Vitamin Kj Vitamin K,

(coagulant (Phylloquinone Vitamin K3
vitamin; anti- Phytomenadione (Menadione
haemorrhagic Phvtonanione) Menaphthone)
vitamin) Vitamin K2
Vitamin C Ascorbic Acid Ascorbic Acid

(anti-scorbutic Dehydroascorbic Sodium Ascorbate
vitamin) Acid Calcium Ascorbate
Vitamin Bj Thiamine Thiamine hydrochloride

(Anti-neuritic (Aneurine) Thiamine mono-nitrate
vitamin; anti- Cocarboxylase
beriberi vitamin) (Thiamine pyro-
phosphate)
Vitamin B, Riboflavin Riboflavin

(Lactoflavin) Sodium Riboflavin-
phosphate
; ;
Vitamin Group Principal Principal

Representatives Commercial Forms
Vitamin B^ Pyridoxine Pyridoxine hydrochloride

(Antiacrodynia (Pyridoxol; Adermin) Pyridoxal- 5 '-mono-
factor; antidermatitis Pyridoxal phosphoric acid ester
factor; rat pellagra- Pyridoxamine (Codecarboxylase)
protective factor)
Vitamin B,2 Cvanocobalamin Cyanocobalamin

(Animal protein Hydroxocobalamin
factor ; anti-pernicious (Aquocobalamin)
anaemia factor;
extrinsic factor;
erythrotin)
Niacin Nicotinic acid Nicotinamide

(Vitamin PP; amide Nicotinic acid
Pellagrapreventive (Nicotinamide
factor; PP factor) niacinamide)
Nicotinic acid (Niacin)
Pantothenic acid Pantothenic acid Calcium-D-pantothenate

(Filtrate factor; Sodium-D-pantothenate
Bios Ila; Bios III; D-Panthenol
Chicken-antidermatitis
factor; vitamin B^)
Biotin d-Biotin d-Biotin

(Vitamin H
Bios II; Bios lib;
Coenzyme R; skin
factor)
Folic Acid Folic acid Folic Acid

(Vitamin Be; (Pteroylglutamic acid)
Vitamin M; eluate Folic acid conjugates
factor; Lacto- (Pteroyl-hexaglutamic
bacillus casei factor) acid ; Pteroyl-diglutamyl-
glutamic acid)
: :
Unlike the nutrients which serve as building materials and storage Vitamins in
substances in the growth of an organism, the vitamins exercise catalytic metabolism
functions. They facilitate the synthesis and degradation of the principle
nutrients, thereby controlling metabolism. Research into these biochemical
processes is still active. The key functions of the vitamins of the B-complex
in particular, have been extensively clarified. Vitamins Bj, Bj, B5, Niacin,
B12, pantothenic acid, folic acid and biotin, and also, in part, their meta-
bolites are incorporated into enzymes which are indispensable for normal
metabolism of carbohvdrate, and protein. In these processes the
lipid
vitamins plav no part as building substances and this also explains why the
daily requirement is small, compared with that of the main nutrients. The
metabolic functions of each vitamin are discussed in detail in Part II of this
book.
If one or more vitamins are either not available at all to an organism, Vitamin
or only in inadequate amounts, certain metabolic processes are impaired, deficiency
leading to disturbances of productivity, growth inhibition and disease. avitaminoses,
Vitamin deficiency also causes disorders of fertility in male and female hypo-
animals as well as increased liability to infectious and parasitic disease. vitaminoses
The functions of the individual vitamins in metabolism are very specific,
so that, in deficiency, one or more defined biochemical reactions in certain
organs can be adversely affected. These disturbances of metabolism can
therefore give rise to very characteristic deficiency symptoms; frequently,
however, the pattern of disturbed health is confused, for example, when
the vitamin is required for several metabolic reactions, or when other
nutrients or active substances are lacking simultaneously.
In countries where diets are unbalanced and inadequate, or where there

are particular dietary customs, certain commonly observed typical disease
patterns have been shown to be due to vitamin deficiency. Knowledge of
the cause has led to the remedy, so that these avitawinoses, which also occurred
quite frequently in Europe even during the last century, are now no longer
of importance. In the developing countries, however, they are still significant
to-dav.
Examples of the most common avitaminoses are

Xerophthalmia, Keratomalacia (Vitamin A-deficiency)
Beri-beri (Vitamin Bj-deficiency)
Pellagra (Niacin-deficiency)
Scurvy (Vitamin C-deficiency)
Rickets (Vitamin D-deficiency)
In addition to the avitaminoses which are characterised by clearly
defined symptoms, there are also bjpovitaminoses - unspecific states - which
are brought about by the inadequate provision of one, or more frequently
Vitamin Bj^ (cyanocobalamin)
several, vitamins. Thev appear in the form of ill-defined symptoms such as
skin changes, reduced vitalitv, lowered resistance to infections, etc.

Finallv, latent hvpovitaminoses are also known; these are states which,
under normal environmental conditions, are not recognisable through
deficiencv svmptoms, but which can immediately induce symptoms under
sudden stress. The relationship between stress and vitamin requirements in
animal maintenance is discussed in detail in the section on "Vitamins and
Carotenoids in Animal Nutrition."
In addition to the hvpovitaminoses and avitaminoses, which are due to Anti-vitamins,

inadequate provision of vitamins, deficiency states occur occasionally vitamin
when the function of a vitamin is disturbed or inhibited. This can occur antagonists
in the presence of specific substances which are designated as antivitamins
or vitamin antagonists. Thev reach the digestive tract as natural dietarv
constituents or as additives (medicaments), either immediately inhibiting
resorption of the vitamin or disturbing its specific biochemical action. Thus,
in raw and dried egg-white, for example, the substance avidin occurs which
forms a complex with biotin in the gastro-intestinal tract, therebv preventing
resorption of this vitamin. Raw fish, especially fresh-water fish, and various
bacteria contain thiaminase, an enzvme that destrovs vitamin B,. Avidin
and thiaminase are destroved bv cooking.
Vitamin antagonists also occur in bracken, various foodplants and in
certain varieties of vegetables. Thus, lucerne contains one or more substances
which markedly reduce the efficacy of the vitamin E present in lucerne,
and significantlv increase its faecal excretion. Antagonists of vitamin E have
also been demonstrated in beans. Linatin - a substance identified in linseed -
has been shown to be an antagonist of vitamin B^,.
The mechanism of action of tvpical antivitamins is based on the similar
chemical structures of the vitamin and antivitamin which enables the latter
to displace the vitamin from its site of action. The antagonistic activity of
certain medicaments mav also be explained in this wav.
Not to be confused with the antivitamins are those nutrient components Nutrient
which, with increasing supplv, raise the requirement for certain vitamins. constituents
This effect can be explained on the basis of the specific function of each that raise
vitamin.When, for example, the enzvmes containing vitamin B^, act mainlv vitamin
on protein metabohsm, it is clear that the vitamin B^, requirement will requirements
increase with increased supplv of protein. Similarly, a large supply of
carbohydrate increases the vitamin Bj requirement.
A somewhat different principle underlies the increase of vitamin E
requirement with increasing supplv of unsaturated fattv acids. In this case
the vitamin E requirement is increased because, in their protection of fatty
acids against oxidation, the tocopherols are themselves inactivated.
Among the substances raising vitamin requirements there are also
The physiological importance of the vitamins for man and animals 13
bacteriostatics (sulphonamides, and coccidiostats) and antibiotics which,

on prolonged administration in high dosage, destroy intestinal flora and
so eliminate a source of vitamins for a host organism.
Diseases, When the defence mechanisms of the body are mobilised and demands
parasites, made on reserve substances there is an associated increase in the activity
bodily stress of enzyme systems. Thus, every increase in metabolic activity leads un-
avoidably to an increased usage of anabolic and catabolic enzymes and
increased vitamin requirement. This is true not only in illness but also for
physical exertion and increased production.
In addition infective organisms and parasites themselves require
vitamins; they compete with their host organism. Intestinal parasites attack
the mucous membranes and interfere with vitamin resorption. Viruses and
bacteria leave toxins in the body, the degradation and excretion of which
require greater enzyme activity.
Vitamin Every individual is constantly exposed to changing influences and

requirements environmental pressures. Therefore the metabolic rate must be adapted
to the immediate necessity, so that vitamin requirements are also subject
to continuous variation between certain limits. It follows that only ap-
proximate quantitative estimates of vitamin requirements can be expressed.
Even in extended animal studies the results are only valid under the special
circumstances of a particular experiment. For example, in studying vitamin
requirements, different results may be expected when different criteria are
followed - e.g. the maintenance of body weight in indoor rearing, or
intensive fattening, work output, milk yield or egg production.
Numerous studies have been made of the vitamin requirements of live-
stock under different conditions, leading to an approximate estimate of
vitamin requirement of the various animal species (see Tables, pp. 34-39).
It is also known under what conditions increases in requirements can be
expected.
In man, however, figures for vitamin requirements cannot be given with
the same reliability as for animals. It is of course possible to determine
minimum amounts which must be supplied daily to avoid severe deficiency
But observations made on domestic animals show that the vitamin
diseases.
maximum production are many times greater than such
requirements for
minimum amounts.
The daily supply of vitamins can only be estimated within certain Hmits
from the contents of a diet. In addition to the external influences already
mentioned, which affect the utilisation of the vitamins supplied, other
factors play a part; the analytical determination of the vitamin contents of
foods is costly and time-consuming, and may be subject to errors of 10%
14 The physiological importance of the vitamins for man and animals
or more, according to the concentrations of the vitamins. Some vitamins

can only be assayed by biological or microbiological methods, which are
also subject to systematic errors.
A further problem in the calculation of the vitamin supply is the degree
of utilisation of the vitamins from different foods and animal feeds. Thus,
for example, ^-carotene and vitamin Bj, as components of food of vegetable
origin, are only incompletely utilised. It assumed that these compounds
is
are bound very firmly to certain parts of the plant-cells and that the en2ymes
of the gastric juice are incapable of liberating them completely in the gastro-
intestinal tract. Accurate data on the degree of utilisation of naturally
occurring vitamins are available in only a few cases.
Consideration of all the factors influencing vitamin requirements leads
to the following conclusions relating to practical human and animal nutri-
tion:
Data on the minimum vitamin requirements are available, based on
numerous experimental results; in view of biological variations and
experimental errors these can, however, only be regarded as approximate
must be taken of appreciable variations
values. In particular cases account
on the one hand the utilisation of administered vitamins is impaired
since,
by many factors and, on the other, vitamin requirements under conditions
of physical stress or increased production can rise to a degree that is
difficult to estimate.
To cover vitamin requirements fully it is therefore necessary to add a

supplement to the diet so that even in unfavourable cases adequate suppUes
are assured.
Contrary to other active substances, such as the hormones, vitamins can
be absorbed in large quantities without ill-effects. Only when the supply
exceeds a certain upper limit (more than a hundred times the requirement
formost vitamins) and, for a prolonged period, can symptoms of so-called
hypervitaminoses develop. So far, hypervitaminosis in man has only been
observed in the case of excessive administration of vitamin D.
Vitamins in Human Medicine
While the vitamins are indispensable as nutritional factors their medicinal Therapeutic
no less important. These embrace
applications for therapeutic purposes are applications
both the elimination of hypovitaminoses which can arise under a variety of vitamins
of conditions, and also the true pharmacodynamic actions of different
vitamins.
In the simplest case hypovitaminoses can result from inadequate or Hypovitaminoses

unbalanced nutrition. In most cases, however, the cause is an increase
above the normal vitamin requirement as a consequence of exceptional
demands on the body such as growth, pregnancy and lactation, infections,
disturbed resorption due to gastro-intestinal diseases, destruction of
intestinal flora by antibiotics, convalescence, increased physical exertion etc.
Such cases are treated by the administration of a single vitamin or a vitamin
mixture.
In replacement therapy of hypovitaminoses the doses generally used Pharmaco-

correspond with the normal daily requirement or a moderate multiple dynamic
thereof. Utilisation of the pharmacodynamic properties of some vitamins, action
however, involves significantly higher dosages quite unrelated to daily
requirements. The pharmacodynamic importance of the vitamins now
covers a wide field of application, illustrated by some examples which
follow.
Vitamin A is used in lowered resistance to infections, lesions of the skin Fat-soluble

and mucosa of different origins, disorders of dark-adaptation and liver vitamins
disorders with diminished storage capabiUty for vitamin A.
The main aim of vitamin D administration is the prophylaxis and therapy
of rickets by normalisation of the metabolism of calcium and phosphate.
Vitamin D finds further application in cases of bone atrophy to enhance
callus formation after fractures and to improve tooth consistency. During
pregnancy and lactation the vitamin D requirement increases.
Arising from its manifold physiological functions, vitamin E has signifi-
cant indications as, for example, in cardiac, vascular and muscular disorders,
and the risks of miscarriage. Therapy with vitamin E is also indicated in
disorders of fat absorption (e.g., fibrocystic disease of the pancreas, sprue)
and when using diets with high levels of unsaturated fatty acids.
Corresponding with the most important points at which vitamin K^
acts in the blood coagulation system, the most important indications are
certain disorders of blood coagulation'which may result, for example, from
prolonged administration of antibiotics or sulphonamides, or from treat-
ment with anti-coagulant drugs.
The areas of application of vitamin C are determined mainly by its Water-soluble

physiological functions. Apart from the elimination of hypovitaminotic vitamins
Vitamin E (acetate of d-a-tocopherol)
Vitamins in human medicine
States resulting from increased demand (increased metabolism resulting

from most important
fever, infections, strenuous physical exertion etc.) the
application is in the supportive treatment and prophylaxis of infections.
Vitamin C is also used to counteract toxic effects of industrial poisons and
drugs, and in cases of poor wound healing and gum bleeding.
Vitamin B^ deficiency leads to severe functional disorders, especially
in the central nervous system and the heart. The indications therefore
comprise, above all, nervous inflammations, neuralgias and cardiac dys-
function, caused for example, by alcoholism.
The therapeutic use of vitamin B^^ is indicated, in particular, in neuro-
logicaland neuromuscular disturbances e.g. cases of alcoholism or persistent
tremor due to Parkinson's disease or to a severe degree of arterial calcifica-
tion. Vitamin B^ is also indicated in conditions of increased tendency to
vomiting and dizziness (e.g. nausea of pregnancy, post-anaesthetic vomiting).
Vitamin B2, nicotinamide and biotin, other constituents of the natural
vitamin B-complex, are widely used together with other vitamins of the
B-group in polyvitamin preparations.
Pantothenic acid is used mainly in the treatment of inflammatory processes,
functional disorders and diseases of skin and mucous membranes. Relaxation
of intestinal muscles, particularly after operations, can also be effected by
pantothenic acid therapy. For medicinal purposes the alcohol of pantothenic
acid (panthenol) is superior to the acid or its salts.
Folic acid and vitamin B^^ ^'^^ closely related in their actions and are
nowadays frequently used together in the treatment of vtirious forms of
anaemia.
Vitamins and Carotenoids in Food
Development Advancing industrialisation in all areas of daily life and the development
of the food of nutritional research during this century have led to changes in human
industry nutrition. The more economic employment of female labour
increased and
in business and industry on the one hand, and the desire for more leisure
on the other, call for a reduction in the time spent on domestic preparation
of meals. Modern nutritional science offers a varied diet of full value in
terms of nutrient and active substance contents with maximum independence
of local and seasonal factors which might influence supplies of vegetable
and animal raw materials. These conditions have led to the growth of a
food industry which not only relieves housewives of part or all of the
preparation of food, but also solves problems of food preservation and
transportation, which can be important to countries in difficult geographical
situations.
The industrial preparation of foods cannot progress without continued
scientific development of suitable processing methods. The losses of
nutrients and active substances occurring in the domestic preparation of
foods are difficult to establish. On the other hand the technology of the
food industry can ensure that raw materials are treated only in suitable ways,
control unfavourable effects of light, air, moisture etc. and compensate for
unavoidable losses by supplementation. In addition the industry can present
food with a satisfactory and appetising appearance, minimising the destruc-
tion of natural colouring agents or supplementing them with natural pig-
ments.
In addition to the adequate production of foodstuffs, three basic pro-
blems present themselves to the food industry of to-day production of foods:
of full value that are both stable and satisfying. In each of these problems the
vitamins and related substances play an important part.
Meeting the As already mentioned, normal function and health require, in addition
vitamin to proteins, fats, carbohydrates and water, different regulators to maintain
requirements metabolism in all its complexity. Among these are the vitamins essential for
life, without which the smooth working of vital processes would be
impossible. The way from this discovery to the isolation of each of the
vitamins and to the quantitative establishment of requirements has been
long and laborious.
To-day, normal requirements of vitamins calculated from scientific data
from different expert committees which are in good general

are available
agreement. Mention may be made of the recommendations issued by the
Food and Nutrition Board, National Research Council, USA (Recommended
Dietary Allowances, eighth revised edition, 1974), the British Medical
Association, the German Society for Nutrition and the Swiss Federal
Department of the Interior (Order of 7. 3. 1957) (see also requirement data
forman detailed in Part II at the end of the chapters on individual vitamins
under the heading "Requirement").
Vitamins and carotenoids in food
Vitamin supplementation levels now adopted by the food industry are

based on these requirement figures, be it that a daily portion of a certain
food contains one third, one half or the total requirement of the vitamins,
be it that another convenient consumer unit is enriched with the respective
quantity of vitamins. In Switzerland, for example, each declaration must
clearly indicate which vitamins and what amounts are present in a definite
portion of a food. Enriched food products are subject to constant control
by state institutions.
The addition of vitamins to food products is generally described in the
literature as "vitaminisation". But, according to the type of food, these
supplements have different aims which can be defined as follows:
Revitamimsation: Compensation for losses in processing, i.e., restoration

of the original naturally present vitamin contents.
Standardisation: Compensation of natural variations.
Enrichment : Addition over and above the initial natural level.
Vitaminisation: Addition of vitamins to foods which represent ideal
vehicles for a particular vitamin, but which do not necessarily contain that
vitamin naturally.
The idea of revitaminisation is that vitamins should be added to those Revitaminisation

foods which have suffered vitamin losses during manufacture, i.e. restora-
tion of the initial content is desirable.
Flour is a typical example of a food product, the revitaminisation of
which is regulated by law in many countries. Wheat is a source of vitamins
which should not be underestimated. As the following Table shows,
however, the vitamin losses in flour are quite considerable even at a milling
extraction rate of 70%.
Vitamin Losses in Flour
\' itamin B, V itamin B. Vitamin B, Niacin
100 g wholemeal contain

(mg) 0, 5 0,•15 0.45 5.0
100 g white floy^: of 70%
extraction rate contain (m g) 0,,1 0,.05 0.08 1.2
The present-day consumer is turning more and more from whole wheat
products to whiter and more readily digestible varieties of bread. Since the
greater part of the vitamins is present in the darker, outer layers of the grain,
which are removed increasingly as the extraction rate falls, white flour is no
longer an important vitamin source. Bread remains, however, a basic food.
'//,
I
Vitamins and carotenoids in food
SO that an important part of the supply of vitamins would be jeopardised

if these losses were not taken into account by revitaminisation in wide
areas of the world. Thus about 250 million people in the west eat bread
enriched with vitamins. The supplement is of the same order as the losses
corresponding to the level of extraction.
The following Table gives examples of the statutory admixture levels
in some countries (additions in mg/kg flour) for flour of SoOq extraction.
Vitamin Additions in fngjkg Flour
Vitamin Bj \'itamin B. Niacin
USA* 6.4 4.0 52.9

Peru 4.0 4.0 30.0
Denmark 5.0 5.0 -
England 2.4 - 16.0
Switzerland** 2.81 1.70 33-67

4.18 2-53 50.0
* The higher levels of the revision 1974 have been considered.

** The upper figures relate to semi-white flours, the lower to white flour.
A similar situation exists in countries where rice is a staple food. Hardly

any vitamins remain in polished rice; for this reason the problem of re-
vitaminising rice has been studied and is now practiced.
Another example of revitaminisation is the addition of the fat-
typical
soluble vitamins and D to skimmed milk, since the naturally occurring
A
vitamins are almost completely removed with the cream.
The vitamin contents of fruit and vegetables vary considerably under Standardisation
the influence of weather, season, soil conditions and other natural factors. and
The consumer would like to be assured, however, that in a half-litre of enrichment
fruitjuice, for example, he has a definite quantity of vitamin C, independent
of season or origin. To satisfy this expectation many countries have legally
regulated the standardisation of the vitamin content of certain food products.
In fact, manufacturers now adjust the vitamin C content of fruit juices, for
example, to or above a definite standard. In this way the general principle

is followed that the vitamins added to a food should be, above all, those that
it already contains naturally.
Seasonal variations of vitamin content are also particularly important
in milk (and therefore also in milk products). The vitamin A content of
milk, for example, is substantially higher during the summer grazing period
than in winter. In some countries it is therefore customary to add vitamin A
to milk during the winter months.
Vitamins and carotenoids in food 23
The question of compensating for vitamin losses arises also in evers*

form oi communal provisioning where problems of preparation, storage, etc.
can frequently be quite considerable. The use of standardised or enriched
foods is particularly appropriate in such cases.
Vitaminisation The principle of vitaminisation can be illustrated bv the example of

margarine. Although originally a substitute for a food, margarine is now
important in modern nutrition. produced mainlv from vegetable oils
It is
and fats and has virtually no natural vitamin A activitv. However, it is

consumed instead of butter rich in vitamin A, and is itself an ideal carrier
for the fat-soluble vitamin A. It was therefore an obvious process to add
vitamin A to margarine to ensure an adequate supplv of this important
active substance to the population. This addition cannot be considered
either as revitaminisation or as standardisation or enrichment; it is a clear
example of a vitaminisation measure.
The following Table lists the quantities of vitamins A and D added to
margarine in various countries, some voluntarily and some statutorilv.
In recent times many varieties of special margarines with high contents
of unsaturated fatty acids have been marketed. On the basis of research
results must be assumed that the human requirement for vitamin E
it
increases with the consumption of unsaturated fatty acids, and thus vitamin E
is now added to such margarines of high dietetic value.
The developing countries are important areas for the application of

vitaminisation. As a result of shortages of food and rapid increases of
population in these areas, new sources of food must be found. Food
products have been developed which can be produced industrially from
cheap, locally available raw materials and which have adequate contents
of proteins of high biological value. Since such products usually contain
only some of the essential vitamins, and mostlv in inadequate amounts,
their vitaminisation is the most practical means of providing the vitamin
requirements of the population.
Stabilisation Many foods are exposed, to varying degrees, to the effects of light, air
of foods and heat during their production, storage and transport. Atmospheric
oxygen, in particular, often causes unpleasant changes in taste and appearance
and also losses of vitamins. Especially in fruit juice drinks, oxidation of
fruit components causes changes in the colour and taste. Ranciditv of fats
is another example of the oxidative degradation of food products.
Substances added to stabilise foods and protect them from oxidation
aretermed antioxidants. Thev have the property of binding the destructive
oxygen before it can attack the food being protected; the antioxidant is
itself destroyed in the process.
In addition to the well-known non-physiological antioxidants (e.g.
BHA, BHT), also the vitamins C and E possess antioxidant properties.
:5c:f^ •=:-.. :«*»^?6§fS'^
Yitamin Supplements to Margarine in IU\kg
Vitamin A Vitamin D
Australia 30,000 4,000

Austria 20,000 1,000
Belgium 20,000 1,000
Brazil 15,,000--50,000 500-2,000
Canada 33,000 -
Chile 14.,000--18,000 1,000
Denmark 24,,000 — 6,000 lU as ^-carotene 5 00
Finland 20,000 2,500-3,500
Germany 20.,000--30,000 1,000
Greece 25,000 1,500
Great Britain 30.,000--33,000 2,900-3,500
Israel 30,000 3,000
Japan 30^,000--40,000
-
Mexico 20,000 2,000
Netherlands 20,000 1,000
Norway 20,000 2,500
Philippines 22,000 1,100
-I- 100 mg vitamin Bj
Portugal 20.,000--35,000 875-1,000
South Africa 20,000 1,000
Sweden 30,000 1,500
Switzerland 30,000 3,000
Turkey 20,000 1,000
u.s.a' 33,000 4,400
The solubility characteristics of these vitamins determine their fields

of application. Vitamin C is water-soluble, vitamin E is fat-soluble; there-
fore vitamin C (ascorbic acid) is suitable for the stabilisation of drinks, for
example, while vitamin E (tocopherol) can be used for fats. Since it is
possible to calculate the amount of oxygen which may be present in a

sealed container, such as a bottle or a jam pot, the necessary amount of
antioxidant can be added accordingly.
Vitamin C is also used as an antioxidant in the treatment of beer and
wine. In the latter case, this permits a physiologically desirable and signifi-
cant reduction in the amount of sulphur dioxide which would otherwise
be necessary.
The vegetable oils (e.g. ground-nut and sunflower oils), which are
known to be more stable than animal fats (lard), show that nature is capable
26 Vitamins and carotenoids in food
of Stabilising its own products. This is the result of the relatively high content
of vitamin E-active compounds (tocopherols) already naturally present in
vegetable oils. If oils and fats are to be additionally stabilised over and
above the degree resulting from the natural antioxidants present, either
additional vitamin E can be added, depending on the original content, or
ascorbyl palmitate, a slightly fat-soluble ester of vitamin C, can be used.
Ascorbvl palmitate and other fatty acid esters of ascorbic acid enhance
the antioxidant action of the tocopherols.
In baking practice, flours with low gluten contents require the addition Baking
of so-called "baking improvers." The non-physiological compounds which improvers
have been used for this purpose, such as potassium bromate, ammonium
persulphate, nitrogen trichloride etc. can be replaced by the equally active
vitamin C. In many countries vitamin C is the only baking improving agent
permitted by food regulations. The quantities added are between 2 and
5
grammes per 100 kg of flour.
Meat and meat products are cured by the addition of salt, nitrate and Curing
nitrite. Curing salts improve and stabilise the colour of the meat. In this
process, the nitric oxide (NO) formed by the reduction of nitrate or
nitrite reacts with the pigment present in muscle to form the more stable
red nitrosomvoglobin. As a reducing agent, sodium ascorbate (or ascorbic

acid) is capable of accelerating this curing process and achieving a uniform
colour. In addition, the use of sodium ascorbate allows a reduction in the
amount of nitrite used. In practice 25-50 grammes of sodium ascorbate are
added to 100 kg of meat. Any residual sodium ascorbate will also provide
some protection against oxidative processes and preserve colour.
Ever food products have been manufactured, they have also been
since Colouring
coloured. The consumer has thus become accustomed to the appearance of of food
certain coloured foods to an extent that in some cases, a food product will products
no longer be bought in a natural colourless form. But nature, too, provides
a rich varietv of colours. The carotenoids form an important group of natural
pigments. They provide colour tones from yellow to red for many foods
such as tomatoes, carrots and oranges. Many carotenoids also possess
provitamin A properties, i.e. they exhibit a degree of vitamin A activity
in thebody; these substances therefore possess, in addition to their purely
pigmenting property, an additional physiological value.

Among the synthetic carotenoids now available, the following are of
special importance: /3-carotene, /?-apo-8'-carotenal (C30), canthaxanthin
and /3-apo-8'-carotenoic acid {C-.^ ethyl ester. The carotenoids are fat-
soluble; thev are therefore suitable for the colouring of margarine and
cheese. In addition, nature has shown that, in many fruits, aqueous systems
can be coloured by exceptionally fine suspensions of these pigments.
Carotenoid formulations have therefore been developed which also allow

the colouring of aqueous solutions. This makes available physiologically
unobjectionable pigments, identical with the natural substances, with which
lemonades, effervescent powders, syrups and fruit juice drinks, as well as
soups, pudding-mixes, sauces etc., can be coloured.

:
Vitamins and Carotenoids

in Animal Nutrition
During the past fifty years, livestock husbandry has undergone profound Production
changes. While free-choice feeding was the rule in the past, the structural increases in
changes in, and continuous growth of, the population to-day call for the livestock
most intensive management possible; this can only be achieved by some management
degree of industrially organised production. Increases of production levels
can be achieved by the following measures
Genetic improvement of stock;
Improved management (housing conditions) and hygiene; and
More intensive and regulated feeding systems.
Considerable success has been achieved in these directions in recent
vears, as shown bv the examples of comparative production in the following
table.
Increase of Animal Production
50 Years To-day Improvement in

ago Yield
Dairy cow Annual milk

production 2,500 kg 4,000 kg 1,500 kg
(=60% more milk)
Frisean herdbook cow Butter-fat content 3-io°o 3-75% 0.65%
(=21% more fat)
Pig Daily live-weight gain 250 g 650 g 400 g ( + 28%)

(i6o°o increase
of weight gain)
Danish meat pig Feed efficiency kg
feed per kg
live-weight gain 4.00 2.50 -1,5 kg (38% less
feed required)
Poultry (laying) Egg production per
bird per year 130 eggs 280 eggs 150 eggs (115 %
increase of laying
performance)
30 years ago To-day Improvement in yield
Poultry (table) 8-week live-weight 400 g 1,400 g 1,000 g (250^0

increase of 8-week
live-weight)
Feed efficiency kg feed
per kg live-weight gain 3.90 2.15 -1.75 (45% less
feed requirement)
:
Vitamins and carotenoids in animal nutrition 29
Deficiencies Animal production formerly depended primarily on feedstuffs available

in earlier on the farm (grass, hay, straw, cereals, root vegetables, whey products,
feeding methods and cereal and domestic residues). These might, in fresh condition, more or
less satisfy animals' requirements for basic nutrients - proteins, fats and
carbohydrates. It is now known, however, that the necessary quantities of

many individual nutrients, especially the mineral salts, trace elements and
the vitamins, may be lacking in such diets. Furthermore, although the
vitamins may be present in adequate amounts in fresh feeds, they suffer
considerable losses during conservation and storage (hay, straw, cereals,
turnips, potatoes etc.). The magnitude of these losses on the farm is
indicated in the following Table showing figures for the destruction of

carotene (provitamin A) at different stages of hay production and storage.
Loss of Carotene during Storage of Hay
Carotene content (mg/kg dry matter)
Freshly cut grass 213 = 100%

Fresh hay 29 = 14%
Hay, stored 13 weeks = 7%
Hay, stored 20 weeks 10 = 5%
Hav, stored 28 weeks 4 = 2%
Thus, when the feeding of livestock was more haphazard, they frequently
and more particularly towards springtime, from vitamin
suffered in winter,
deficiencies, resulting in low yields, unsatisfactory fertility, litters of feeble
young, increased incidence of diseases and deaths from unknown causes.
Improvement of animal nutrition therefore involves not only the pro-
vision of a feed of better quality in respect of protein, fat and carbohydrate
but, above all, the supply of adequate and known levels of the mineral
salts, trace elements and vitamins.
Vitamin Regular provision of vitamins therefore has an established place in

supplements modern animal nutrition. This is one of the direct results of the changes in
feeding and management conditions of livestock, which have accompanied
increasing intensification. The important factors that have influenced the
provision of vitamins for animals have been the following
Restriction of free choice of feed. Under increasingly intensive husbandry,
stock formerly on free range no longer receive adequate amounts of
provitamin A from green forages. In the absence of sufficient green feeds,
the balance of the requirement must be restored by supplements of vitamin A
in the diet.
I ^
^
1-ÎVV XI
^'^{^^,)i^ ..'rti
1
Growing The use of compound feeds is especially-

use of manufactured feeds.
prevalent in modern poultry management and also plays an increasing role

in the management of pigs and ruminants. The raw materials used for
industrially manufactured feeds contain hardly any carotene, and their
vitamin contents are seldom adequate. Modern compounds are therefore
usually enriched with vitamins. Supplements currently used are:
In cattle feeds (for rearing, milk production and fattening): vitamin A,
vitamin D,, vitamin E.
In milk-replacers for calves: vitamin A, vitamin D-, vitamin E, vitamin C,
vitamins of the B-complex.
In pig feeds: vitamin A, vitamin D3, vitamin E, vitamin K, pantothenic
acid, vitamin B,, nicotinic acid, vitamin B,2, vitamin B^, and occasionally,
biotin.
In milk-replacers for piglets: vitamin A, vitamin D„ vitamin E, vitamin K,
vitamin B^, vitamin B5, nicotinic acid, pantothenic acid, vitamin Bj^,
vitamin C; occasionallv vitamin B, and biotin.
\n poultry feeds: vitamin A, vitamin D,, vitamin E, vitamin K, panto-
thenic acid, nicotinic acid, folic acid, vitamin B,, vitamin B,, and, more
recently, vitamin Bj, vitamin B^,, vitamin C, and biotin.
Lower feed consumption per unit of production. Selection in favour of eco-
nomic production strains results in animals giving considerably higher
absolute yields than formerly for only slightly increased intakes of feed,
so that the production unit (kg milk, meat, eggs etc.) is attained on signifi-
cantly less feed; therefore animals receive relatively less of the vitamins
occurring naturally in a ration. Modern compounds are also characterised
by increased nutrient density, the utilisation of which requires increased
amounts of the vitamins involved in metabolism (mainly those of the B-
complex). These facts have necessitated the increased vitaminisation of
mixed feeds and supplements.
Increased turn-over of nutrients: Increasingly higher yields necessitate the
more rapid turn-over of nutrients, which also leads to the increased con-
sumption of those vitamins directly involved in metabolism, especially
those of the B-complex.
Influence of stress:Modern management of livestock imposes increasing
"stresses" on animals. The best possible utilisation of housing space,
artificial lighting, ventilation and hygienic measures is necessary for eco-
nomic production. These conditions, however, generate certain agitations

and anxiety states which, together with fighting for food and the "pecking
order", act as stresses. The adequate provision of vitamins assists animals
to stand up to these strains.
Increased risk of infections: The concentration of animals in large herds,
and also their transport over distances - of chickens and calves for example -
increase the risk of infections and parasitic attacks (e.g. coccidiosis in
poultry). Relationships between an ample supply of vitamins (particularly
32 Vitamins and carotenoids in animal nutrition
of vitamins A, K and the B-complex), and increased resistance to parasitic

attack and infectious disease, have been demonstrated experimentally, as
has the enhancement of antibody formation by vitamin A. In practice these
risks can be countered by periodic supplements of vitamins either via the
drinking water or by injection.
Administration of drugs: The danger of infestation of large herds with
chemo-
entero-parasites has led to the administration of drugs (antibiotics,
therapeutics) becoming These drugs are also admixed
a regular operation.
with feeds so that they are ingested continuously by stock. However, such
medication itself can cause stress, resulting in increased vitamin require-
ments.
In addition, some of the preparations used for the mass-treatment of
animals have antagonistic effects on some vitamins of the B-complex and
are to be considered in the nature of antivitamins. To compensate for
vitamin losses resulting from such drug administration, the vitamins con-
cerned must be added in increased amounts in either the feed or via the
drinking water.
Increased vitamin supplies also enhance the development of immunity,
so reducing the risks of re-infection during medication.
In allof these ways, the vitamins are capable of promoting the health,
fertility and productivity of intensively managed livestock.
The daily vitamin requirements of different animal species have been Vitamin
the subject of numerous experimental studies and practical investigations requirements
in recent years. The figures summarised in the following Tables are based of livestock
on data from many publications and represent vitamin levels recommended
for the total supply of biologically available vitamins for the respective
types of livestock, taking into account their requirements and varying
environmental and feed supply conditions.
Vitamin A (vitamin Aj [-alcohol])
Recommended Vîtamin Levels for Poultry

Amounts per kg feed (dry matter)
Chicks and Chicks and Hens Turkey Turkey Turkey

Broilers Broilers (laying and (starting) (growing (breeding)
(starting) (growing) breeding) and
fattening)
Vitamin A lU 15,000 10,,000 10.,000 15:,000 12,000 12,000

20,000 IJ:,000 IJ ,000 20 ,000 16,000 16,000
Vitamin D3 lU 1,500 I.,200 2.,000 I, ,500 1,500 1,500
2,000 2,,000 3:,000 2,,000 2,000 2,000
Vitamin E lU 30 25 50 35 30 40
60 JO 60 70 60 ^0
Vitamin K, mg 3
2 2 3 3 3
8 8 8 12 8 70
Vitamin Bj mg 3 3 3 5 3 3
Vitamin B, mg 8 6 6 8 6 8
Nicotinic aaid mg 50 40 40 80 70 70
Pantothenic aciid mg 20 12 15 20 15 25
Vitamin B^ mg 7 5 5 7 5 6
Vitamin B,^ mg 0.030 0.020 0.015 0.020 0.015 0.020
Folic acid mg 1-5 1.2 1-5 1-5 1-5 2.0
Biotin mg 0.15 0.15 0.20 0.35 0.20 0.30
Choline mg 1,500 I.,300 I,,100 2.,000 1,700 1,700
Vitamin C mg /;o 60 200 IJO IJO 200
The vitamin requirements of ducks may be taken to be of the same order as the requirements of hens.
Figures in italics refer to unfavourable environmental conditions, high production and/or marginal composition
of the feed.
Recommended Vitamin Levels for Pigs

Amounts per kg feed (dry matter)
Piglets* Piglets Pigs Pigs Sows

(early weaned) (starting) (growing) (fattening) (breeding)
Vitamin A lU 4°:,000 20,,000 10.,000 5.,000 12 ,000

60.,,000
P:,000 12 ,000 8:,000 20,,00(9
Vitamin D lU 4:,000 2.,000 I, ,200 I.,000 I, ,200

6:,000 i ,000 /, JOG 7.,200 2 ,000
Vitamin E lU 50 30 25 20 25
100 80 JO 40
Vitamin K3 mg 3 z I I 2
8 6 /
Vitamin B, mg 6 3 2.5 2.0 2.5
Vitamin B2 mg 8 6 5 4 6
Nicotinic acid mg 35 25 20 15 20
Pantothenic acid mg 25 20 15 13 T2
Vitamin B,, mg 8 6 5 4 5
Vitamin B12 mg 0.06 0.06 0.03 0.02 0.02

Folic acid mg I.O 0.8 0.5 2.0
Biotin mg 0.25 0.25 0.15 O.IO 0.22
Choline mg I.,400 I, ,200 I. ,000 900 900
Vitamin C mg /oo ^00 100 100 100
* calculated on the base of milk-replacer

of the feed.
Recommended Vitamin Levels for Cattle, Sheep and Goats

Amounts per animal per day, for calves per kg feed (dry matter)
Calves Cattle Cattle Dairy Cows Sheep and

0-5 months (rearing) (fattening) Goats
Vitamin A lU 40,000 25,000 40,000 50,000 4,000

60,000 40,000 60,000 100,000 10,000
Vitamin D lU 4,000 3,000 5,000 8,000 250
6,000 J,000 6,000 I J,000 JOO
Vitamin E lU 50 150 250 350 25
100 ^00 J40 1,000 JO
Vitamin K, mg 3
/
Vitamin B, mg 3
8
Vitamin B2 mg 10
Nicotinic acid mg 20
30
Pantothenic acidimg 12
Vitamin B^ mg 4
S
Vitamin Bj^ mg 0.02
O.OJ
Folic acid mg 0.5
I.O
Biotin mg 0.15
*
0.2J
Choline mg 400
Vitamin C mg JOO
of the feed.
/ /*»
Mm^%
Vitamins and carotenoids in animal nutrition
Recommended Vitamin Levels for Horses

Amounts per loo kg live-weight
Foals, Weaning Working and Race Horses and

(Yearlings) Saddle Horses Breeding Horses
Vitamin A lU 10,000 10,000 15 ,000

18,000 18,000 20 ,000
Vitamin D lU 1,000 1,000 I ,400
2,000 2,000 2 ,400
Vitamin E lU 50 50 200
90 90 JOO
Vitamin K, mg 5
10 + +
Vitamin Bj mg 15 15 20
Vitamin B2 mg 15 15 20
Nicotinic acid mg 25 25 25
Pantothenic acid mg 15 15 15
Vitamin B^ mg 10 10 15
Vitamin B12 mg 0.17 0.17 0.25
Folic acid mg 10 10 10
Biotin mg O.IO
Choline mg 200 200 250
Vitamin C mg 2 JO 2J0
of the feed.
Recommended Vitamin Levels for Dogs, Cats, Rabbits, Fur-Bearing Animals and Fish
Amounts per kg feed (drv matter)
Dogs Cats Rabbits Mink and Carp and Trout and Eel''
Pet Pet Foxes other other

Laboratory Laboratory Cyprinids'' Salmonides
Vitamin A lU 8 ,ooo 30 ,000 10,000 10,000 8,000 15 ,000 12,000

12 ,000 40 ,000
Vitamin D lU 8oo I ,500 1,000 1,000 1,800 3 ,000 2,000
I ,200 2 ,000
Vitamin E lU lOO 200 40 80 40 80 160
IJO 400
Vitamin K, mg lO 10 3 8 4
10 20
Vitamin B, mg 3 10 2 4 6 15 50
Vitamin B, mg 5 8 6 6 25 30 60
10 10
Nicotinic
acid mg 20 60 50 30 70 180 80
2J 100
Pantothenic
acid mg 6 20 20 15 60 50 60
10 JO
Vitamin B^, mg 5 5 2 2 6 15 20
IJ
Vitamin B,, mg 0.03 0.02 0. 01 0.03 O.OI 0.05 0.15
O.OJ O.OJ
Folic acid mg 0.3 0.5 0.6 1.0 5.0 5.0
O.J I.O
Biotin mg 0.25 0.20 0. 20 0.25 0.30 2.50 0.80
O.JO O.JO
Choline mg I ,000 I ,500 1,300 1,000 800 I ,800 800
I ,J00 / ,J00
Vitamin C mg IJO IJO 100 150 500 300

Inositol mg 200 150 I ,000 150
200
* for fr\- and parental tish before and after laying the vitamin level should be increased by
50°o-
** for elver (smaller than
4 g) all vitamins except A and D
should be increased 3 times, for fingerhng (4-10 g)
2 times.
Figures initalics refer to unfavourable environmental conditions (laboratory animals under experimental stress),
high production and/or marginal composition of the feed.
:
Technological Properties and Formulations

of Vitamins
The application of vitamins in pharmaceuticals and for the fortification

of food and animal feeds can of course be achieved to a degree with vitamin-
rich natural materials such as yeast, wheat-germ etc., or with concentrates
or extracts prepared from such products. These, however, are now seldom
able to meet the critical requirements of the modern processing industries.
Most of such materials have rather low and also varying contents of the
vitamins, and they often contain additional substances which adversely
affect the organoleptic properties and storage stability of the products
in which they are included; further, they frequently occur in forms un-
suitable for many applications.
Advances in organic chemistry and the development of new techniques
have now made possible the economic synthesis of most vitamins on an
industrial scale, and the use of synthetic vitamins is now the predominant
practice in human and animal nutrition as well as in medicinal products.
Many experimental studies have shown that the synthetic compounds are
identical in all biological properties with the naturally occurring vitamins,
and the identit)' of the activity of the synthetic and the corresponding
natural vitamins has been well established.
The industrial production of vitamins with a high degree of purity, Application

has largely solved the organoleptic problem as well as the difficulty of forms
providing accurate, known quantities. A further problem, however, was
that many vitamins are very sensitive substances, unstable in adverse
environments, so that the development of more stable forms became
necessary for their extended application in the food and animal feed
industries. Certain other properties of some pure vitamins, such as their
solubility, physical state, concentration etc., also restrict their usage
possibilities, so that special forms had to be created, more suitable for speci-
fic applications.
Formulations of the vitamins suitable for the most varied purposes
can be prepared by the following methods
The synthesis of stable derivatives.
The addition of stabilisers (antioxidants).
Standardisation with suitable fillers.
Coating with suitable carrier substances.

The transformation of water-soluble vitamins into fat-soluble derivatives.
The transformation of fat-soluble vitamins into water-soluble derivatives
or water-dispersible formulations.
All these methods are used in the manufacture of commercial vitamin
formulations. The simultaneous use of several of these procedures is often
involved, the method of choice being determined by the desired relationship
between physical properties and biological activity.
Technological properties and formulations of the vitamins 41
The most important properties relevant to the use of the various

vitamins, and the usual commercial forms, are as follows.
Fat-soluble Vitamin A is extremely sensitive to oxidation. Its destruction bv

vitamins atmospheric oxygen is accelerated catalytically by light, especiallv bv ultra-
violet light, and also by metal salts, peroxides, and heat, particularlv in the
presence of moisture. Destruction of the vitamin is also facilitated bv the
finely divided state which is necessary to ensure homogeneitv and optimal
absorption.
The instability of vitamin A-alcohol, the essential form of vitamin A,
has led to the industrial preparation of its somewhat more stable esters,
the acetate and palmitate. Additional stability' can be achieved bv dissolving
these in vegetable oils; and further stabilisation obtained bv the addition
of anti-oxidants, which can also be combined with svnergists and complexing
agents. Such forms of vitamin A can be used directlv in fats and oils.
The oily forms of vitamin A however, are unsuitable for processing in
dry preparations such as animal feeds. For such purposes drv powder
preparations have been developed, in which the vitamin A is deposited
in a carrier substance. The most important carrier for stabilising vitamin A
is gelatine, in which the vitamin A must be present as extremely finely
divided droplets to ensure rapid absorption. The particle size of such pow-
ders should be bervt'een 150 and 500 ,« (diameter), and the concentration
approx. 500,000 lU vitamin A per gramme, in order to ensure satisfactorv
dispersion in compound feeds and similar products.
The criteria of particle size and concentration necessitate compromise
solutions. Larger particles, having relativelv smaller surface areas, are more
stable, but they result in more irregular distribution of the vitamin A
in a mix. Smaller particles are less stable because of their relatively larger
surface areas. Highly concentrated powders are thus unfavourable for the
distribution of vitamin A in a feed ; on the other hand lower concentrations
lead to increased costs.
In addition to these drv powder forms, liquid water-miscible formulations
have also been developed. The use of appropriate emulsifiers produces
aqueous dispersions which are suitable for the preparation of solutions and
syrups for human medicine, and for veterinary use as injections and for the
enrichment of drinking water.
\'itamin D (D, and D,) is and
also sensitive to oxidising agents, light,
acids. Since the considerations of stabiHtv and application for vitamin D
closely resemble those for vitamin A, similar commercial forms have been
developed (oily solutions, stabiHsed powders and aqueous dispersions).
Vitamin E
is used mainlv in the form of the relativelv stable a-tocophervl
acetate, since the unesterified tocopherols are rapidlv oxidised and darkened
by atmospheric oxygen. In the presence of moisture the vitamin E-ester
is hydrolysed by acids or alkahs. Vitamin E is also therefore processed to
Technological properties and formulations of the vitamins 45
more stable forms, and adsorbates, granulates and water-miscible prepara-

tions are now commercially available.
Vitamin K^ is slowly degraded by atmospheric oxygen, but is very
rapidly affected bv light and alkalis ; it is however, relatively stable to heat.
Water-soluble The water-soluble vitamins are generally stable in the pure state. In
vitamins aqueous solution, however, they are more sensitive to a number of factors.
Thiamine hydrochloride, the most important commercial form of
vitamin B^, is stable if protected from light and moisture. In aqueous solution
the stability of vitamin Bj is markedly pH-dependent.
Stability is optimal at pH 5.0, and is still good at pH 4.5. In neutral or
alkaline solution however, particularly in the presence of oxidising or
reducing agents, or if heated, vitamin Bj is unstable and is converted to
inactive compounds; heavy metals accelerate this destruction.
In the presence of vitamin B,, vitamin Bj is oxidised in aqueous solution
to thiochrome. This process is facilitated by increasing concentration of
vitamin B, and by the presence of atmospheric oxygen.
In dry preparations the degree of humidity is important for stability.
When conditions are such that the hydrolysis and oxidative decomposition
of thiamine are likely, it is advantageous to use the less sensitive thiamine
mononitrate instead of the more usual hydrochloride. Thiamine mono-
nitrate is forms to improve its stability, and to
also prepared in coated
reduce odour and taste.
its
Vitamin B^ (Riboflavin) is unstable to powerful reducing agents, alkaUs

and light. It is only sparingly soluble in water; to increase the solubility
of riboflavin, solubiHsers such as nicotinamide or salicylic acid are used.
Optimal stability of solutions is at pH 5.5-4.0.

5
The sodium salt of riboflavin- '-phosphate is considerably more water-
soluble. Its stability characteristics are similar to those of riboflavin, but it
reacts with heavy metal ions and especially with calcium to form insoluble
metal salts; the addition of chelating agents can prevent this reaction.
In aqueous solutions, riboflavin acts as an oxidising agent to vitamins Bj,
C and folic acid. It also acts as a photosensitiser and hydrogen acceptor in
the light-induced oxidation of folic acid and vitamin C.
Riboflavin has an unpleasant, lasting bitter taste, but dry preparations
of this vitamin are also available in coated form.
Vitamin B(, (Pyridoxine) in the usual commercial form of the hydro-
chloride is generally stable to heat and oxygen. It is degraded by light in
neutral or alkaline solutions, and to a lesser degree in acid solution; optimal
stability is in the region of pH 3.0-5.0.
A coated preparation of pyridoxine hydrochloride is also commercially
available for special applications.
Pure crystalline vitamin B^^ (cyanocobalamin) is relatively stable to air
in the dry state and in neutral to weakly acid solutions; optimal stability
44 Technological properties and formulations of the vitamins
is at pH 4.5-5.0. Destruction of vitamin Bjj only occurs at elevated tem-

peratures, but this destruction is increased in the presence of vitamin Bj
and, more particularly, nicotinamide. In solution vitamin B12 is also sensitive
to light, especially ultraviolet light. Vitamin C and its oxidation products
(e.g. dehydroascorbic acid), in the presence of copper, manganese, molvb-
date or fluoride ions, as well as alkalis or reducing agents, degrade vitamin
B12. Vitamins B,, B^ and panthenol, however, are compatible with vitamin
Biz-
Dry powder dilutions containing from 0.05 °o to i.o°o of vitamin B12
on a base of, e.g. mannitol or dicalcium phosphate; stabilised adducts on
ion-exchangers and preparations with a gelatine base are
; now commercially
available. For animal feed supplementation, fermentation concentrates are
commonly used; these have contents of up to i per cent vitamin B,2.
Biotin is stable to oxygen, daylight and heat in the dry crystalline state.
Ultraviolet light or powerful oxidising agents can destroy biotin. In strongly
acid or alkaline solutions its biological activity falls rapidly. For the enrich-
ment of animal feeds, a diluted product with a standardised content of i %
biotin is available.
Pure, crystalline folic acid and heat, but is degraded bv
is stable to air
light, especially ultra-violet light. It is most stable in neutral to weakly
alkaline media. It is destroyed by acids, strong alkalis, metal salts and by
reducing and oxidising agents. Vitamin B, is slightly - and vitamin B2
markedly - destructive to folic acid. Panthenol, nicotinamide and vitamin B^
however, are compatible with foHc acid.
Nicotinic acid amide, in the pure anhydrous form and also in aqueous
solution, is stable to air, daylight and heat. Ultraviolet light slowly destroys
nicotinamide. Strong acids, strong alkalis and also some heavy metals reduce
its biological activity.
Nicotinamide is also available in a coated form.
D-Pantothenic acid, as a compound, is very sensitive to many factors, and
it is therefore produced commercially as the calcium and sodium salts
which possess good stability providing moisture is excluded.

Aqueous solutions of pantothenic acid salts are only stable to a degree
at pH 5.0-7.0; they are sensitive to heat and tend to hydrolvse, especially
in the presence of acids and alkalis.
For liquid preparations, D-Panthenol (the alcohol corresponding to

D-Pantothenic acid) - has been developed; its aqueous solutions at pH 4.0-
7.0 are significantly more stable and can be heat-sterilised.
Crystalline \îtamin C (ascorbic acid) is relatively stable in air under
anhydrous conditions the sodium ; salt however, tends to turn yellow with
time. Aqueous solutions of ascorbic acid are sensitive to oxidising agents,
decomposition being accelerated by alkalis and traces of heavy metal ions
minimise oxidation, solutions are treated with metal

(particularly Cu); to
(Cu)-complexing agents at a pH below 6.0. In multi-vitamin solutions and
Vitamin Bj (thiamine hydrochloride)
46 Technological properties and formulations of the vitamins
syrups the stability of ascorbic acid falls as the content of water present
increases. The vitamins and nicotinamide adversely affect the stability
Bj, B2
of ascorbic acid, but thiamine is compatible. Vitamin Bj absorbs blue light
and, in the presence of air, can catalyse the photo-oxidation of ascorbic
acid. The destructive effects of vitamin C and vitamin B2 are reciprocal;
vitamin C is also destructive to folic acid and vitamin B12.
Ascorbic acid is also available commercially as its sodium and calcium

salts and in coated preparations.
Part II: The Individual Vitamins
Vitamin A
Vitamin A (Retinol, Axerophthol) is, in the strict sense, an alcohol,
but it occurs in nature mainly as a fattv acid ester. The highest naturally
occurring levels of vitamin A content are found in certain fish liver-oils
(cod, shark, tunny). Mammalian liver, other meat offals, egg-yolk, milk and
milk products are important foods providing vitamin A to meet human
dietary requirements.
The molecular structure of vitamin A indicates sixteen theoretically
possible isomers, six of which are known. However, only two isomers are
of practical importance, namely all-trans-vitamin A, the form with highest
biological activity, and the 13-cis isomer (also called neo-vitamin A) with a
relative biological activity of 75 °o- Natural vitamin A preparations usuallv
contain about one third neo-vitamin A, while good synthetic products
generally contain considerably less.
The principal commercial forms in use are vitamin A acetate and vitamin
A palmitate which are marketed as oilv solutions, stabilised powders or
aqueous emulsions.
Physico-chemical properties
Structural formula CH3 cH;
Acetate: R^COCH,
Palmitate: R=CO(CH,)i4 CH3
Empirical formula Acetate: C^^H-jO,

Palmitate: C36H60O2
Molecular weight Acetate: 328.5

Palmitate: 524.9
Description Acetate : Bright vellow crystalline powder

Palmitate : Yellow oil, or crystalline mass
Solubility Insoluble in water, soluble in alcohol, readily

soluble in ether, chloroform, acetone and fats and
oils.
JO Vitamin A
Melting point Acetate :

5 7-60 °C
Palmitate: 28-29 °C
Absorption spectrum Vitamin A esters show a characteristic absorption

spectrum, the position of the maximum depending
on the solvent; in isopropanol it is at 526 nm, in
cyclohexane at 328 nm.
Stability Vitamin A alcohol and its esters are rapidly

destroyed bv light, oxygen and acids. They must
therefore be stored in hermetically sealed opaque
containers from which the air has been displaced
by an inert gas (e.g. nitrogen).
Methods of Determining lîtamin A in Simple Mixtures
1. Measurement of U. V. light absorption

This method can only be carried out with pure vitamin A preparations
or simple concentrate mixtures containing no substances with interfering
or irrelevant absorption.
2. Measurement of fluorescence
The method is simple, sensitive and highly specific. Interfering fluores-
cence and quenching mav occur. Therefore, in general, some purification
steps are needed.
j. Carr-Price reaction
Antimony trichloride in chloroform or ethylene dichloride gives a
characteristic unstable blue colour with vitamin A alcohol and its esters.
The reaction is very sensitive and is only shown by a few other substances
which, however, often occur together with vitamin A (polyenes such as
/5-carotene). Cis- and trans-isomers of vitamin A are not differentiated in
this reaction, both giving colours of equal intensity.
4. Anhj'dro method
Vitamin A alcohol is converted by acids into anhydro vitamin A. The
method is highly specific but less sensitive than the Carr-Price reaction.
/. Other reactions
Vitamin A reacts with activated glycerol dichlorohydrin producing
a mauve colouration ; sensitivity and specificity are similar to those of the
Carr-Price reaction. Other suitable reagents are trifluoroacetic acid, tri-
chloroacetic acid or acetyl chloride iron (III) chloride.

:
Vitamin A 5 I
Methods of Determination in Complex Mixtures

(Foods, Feedstuffs, etc.)
The analytical procedure can be separated into the following steps

1. Conversion of vitamin A esters to vitamin A alcohol by alkaline hydro-
lysis.
2. A alcohol from the saponification mixture.

Extraction of the vitamin
3. Chromatographic purification of the vitamin A extract.
4. Determination of the vitamin A content bv fluorescence spectrophoto-
metrv, bv the Carr-Price reaction or by the anhydro method.
In many cases, it is advantageous to extract the active vitamin A substance

from the analytical sample with Hpid solvents before saponification.
For the purification of the vitamin A extract column chromatography on
aluminium oxide is most frequently used, but other adsorbents, such as
calcium phosphate, are also suitable.
Procedures for paper, thin-layer and partition chromatography have
also been described. If the vitamin A content is to be determined by U.V.
spectrophotometry, the irrelevant absorption can be eliminated bv a cal-
culation (Morton-Stubbs correction) which is based on the assumption

that the irrelevant absorption is linear between 310 and 340 nm. In general,
more accurate results are achieved by purifying vitamin A containing
extracts than by applying the Morton-Stubbs correction.
Biological Units and Standardisation
An International Unit corresponds to the activity of 0.344 meg of pure

crystalline vitamin A acetate. The vitamin A Unit of the U.S. Pharmacopoeia
(U.S. P. Unit) is the same as the International Unit. A standard preparation
is supplied as a reference substance by the U.S. P. Reference Standards
Committee (U.S. P. Reference Standards, 46 Park Avenue, New York,
N.Y. 10016).
Units no longer in current use are:

I Cod-liver-oil (CLO) Unit = 550 lU
I Blue-value Unit = 53 lU
I Carr-Price Unit = 33 lU
I Lovibond Unit = 6.6 lU
I Sherman Unit = 0.66-0.8 lU
I Rat Unit = 0.66-0.8 lU
The curative rat-growth test as described for example in U.S. P. XIV is
mostly used for the biological standardisation of vitamin A in pharmaceutical

)
52 Vitamin A
preparations as well as in foods and feedstuffs. This somewhat time-

consuming method can be replaced by shorter procedures such as the
vaginal cornification and Hver storage tests, provided they are shown to
give results in agreement with those of the rat-growth test.
In the assessment of the vitamin A contents of feedstuffs, in addition

to the actual vitamin A activity, absorbability and stability are of decisive
importance. Absorbable, stabilised dry preparations must therefore be used
as standards in the biological tests. In addition, the animal experiments should
be carried out under conditions most closely resembling those occurring
in practice (e.g. chick-growth or chick liver-storage tests).
Physiological Functions
Vitamin A (all-trans-retinol) has an important role in the visual process.

The alcohol is oxidised enzvmaticallv in an oxidation-reduction equilibrium
system to the aldehyde (all-trans), formerly known as retinene but now
called retinaldehvde or retinal. The all-trans-retinaldehyde is isomerised
to the I which combines with the protein opsin to form the visual
i-cis-form
pigment, rhodopsin, the photo-receptor for vision at low light intensities.
As shown in the diagram below, when light falls on the retina - the outer
segments of the rods - the pigment complex is broken down through the
formation of all-trans-retinaldehyde which is not bound by opsin. Ion
transport and membrane potentials are affected by this decomposition which
causes the transmission of an impulse up the optic nerve.
The all-trans-retinaldehyde is isomerised back to ii-cis-retinaldehyde,
which recombines with opsin, regenerating the visual pigment and thus
continually renewing the light-sensitivity of the retina.
Rhodopsin
( 1 1- cis-Retinaldehyde - opsin complex
Light
Retinaldehvde
Opsin + ii-cis- -ii^ all-trans-Retinaldehyde
Retinaldchyde isomerase
+ Opsin
K \
Redn. Oxidn.
M M
ii-cis-Retinol all-trans-Retinol
Vitamin A 53
Because of this essential function of vitamin A in vision a deficiency

leads to impairment of the regeneration of visual pigment which shows
itself as night blindness. This is one of the first symptoms of vitamin A
deficiency.
Vitamin A is also involved in the process of formation and preservation
of function of epithelial tissues and mucous membranes. Lack of vitamin A
therefore leads to a degeneration and keratinisation of epithelial cells of the
ovaries, vagina, testes and cornea. The injury of the cornea leads to the
most widely recognised symptom of vitamin A deficiency, xerophthalmia.
Epithelial tissue injury also decreases its resistance to bacterial invasion.
In contrast to the well understood mechanism of the function of vitamin
A in visual processes, its functions at the molecular level of cell physiology
and its metabolism bodv tissues are, at present, still obscure. Vitamin A
in
appears to have effects on subcellular systems such as the structure of cell

membranes and cell particles as well as on certain enzymes responsible for
cell metabolism and on the biosyntheses and interactions of steroid hormones.
The absorption of vitamin A is greatly enhanced by the presence of

emulsifiers (e.g. bile salts). Vitamin A-esters in food are cleaved before
absorption by a hydrolase in the pancreatic juice, but the liberated vitamin
A alcohol is re-esterified with long-chain fatty acids, especially palmitic
acid, during absorption in the intestinal wall.
General Deficiency Sjniptotus
The symptoms of vitamin A deficiency can be summarised under the

following headings:
1. Symptoms ascribable to damage of the normal visual process.

2. Symptoms of changes in, or functional disturbances of, the skin or
mucosa ("epithelial-protective" function of vitamin A).
3. Symptoms brought about by disturbed bone growth.
4. Symptoms caused by impaired food utilisation, e.g. slow growth and
weight losses.
Individual symptoms may or may not be highly pronounced, and can

vary from individual to individual, from one animal species to another,
and a particular symptom may be outstanding or masked by others, de-
pending on the specific nutritional conditions and other circumstances.
In man vitamin A deficiency leads to so-called night blindn ess a nd, in
severe cases, may result in clouding and abscess formation in the cornea,
as well a s dry skin, hyperkeratosis and loss of hairsheen. Finally, certain
forms of anaemia are known, which are due to an inadequate provision of
vitamin A. A vitamin A deficiency frequently results in loss of appetite,
54 Vitamin A
\'itamin A defi-
ciency in the
chicken: Hyper-
keratosis of mucous
membranes of mouth
and oesophagus.
Jncreased sus ceptibility to infections and parasitic diseases and reduced

ability to combat various forms of stress.
Deficiency Symptoms in Animals
General condition Reduced appetite, growth inhibition, weight loss

and death (all species).
Skin Rough hair or plumage (cattle, pigs, poultry).
Dryness of the horn of the hoof (horse).

Poor wool production, both qualitatively and
quantitatively (sheep).
Crossed beak formation (chicken).
Eves Night blindness, retinal degeneration (cattle,
sheep).
Hyperkeratosis of the cornea, tears (cattle, horse,
rabbit, chicken).
Papilloedema and constriction of the optic nerve
(cattle, dog).
Blindness (cattle, pig, dog).
Vitamin A 55
Visceral gout in the

chicken, a symptom
of vitamin A
deficiency.
The arrows show
clearly visible
areas of pin-point
degeneration.
Malformations in
new-born piglet
due to deficiency
of vitamin A in
the diet of the sow.
:
56 Vitamin A
Nervous svstem Uncoordinated movements (cattle, pig).
Convulsions (pig, cattle).

Paresis (cattle, pig, rat).
Areas of pin-point degeneration in the brain
(chicken).
Raised cerebrospinal fluid pressure, hydrocephalus
(cattle, pig, rabbit, chicken).
Herniation of the spinal cord (pig).
Nerve degeneration (cattle, pig, dog, rabbit, rat,
chicken).
Bones Metaplasias and formation of spongy bone tissue

(cattle, dog, chicken).
Endocrine glands Pituitary and suprarenal cysts (cattle).
Respiratory tract Metaplasia of nasal mucous membrane (chicken).

Increased incidence of infections of respiratory
tract and lungs (cattle, pig, rat).
Alimentary tract Keratinisation of mucous membranes of mouth,

oesophagus and salivary ducts. Sensitivity to
bacterial infections and parasitic attack.
Liver Metaplasia of bile ducts, degeneration of Kupffer

cells (rat).
Urinarv tract Increase in urinarv calculi (cattle, sheep, rat).
Nephritis (chicken).
Reproductive system Male animals

Reduced sexual activity, degeneration of seminal
epithelium, reduction in number and motility of
sperms, numerous abnormal spermatozoa, atrophy
of testicles and accessory sex glands (cattle, sheep,
dog).
Female animals:
Metaplastic hyperkeratosis of mucous membranes
of the vagina (rat), cervix (cattle) and uterus
(guinea-pig).
Increased infections of these mucous membranes
(cattle, pig).
Ovarian atrophy, decrease in ovulation and

fertility, disorders of oestrus cycle (cattle, pig).
Vitamin A 57
Fundus of the eye

with point of entry
of optic nerve and
surrounding tissue
of a healthy calf.
Papilloedema of the
optic disc - a
diagnosis of vit-
amin A deficiency
in the calf.
;
Vitamin A
Blindness in calf
due to vitamin A
deficiency induced
by a poor diet.
Eye changes are
especially pro-
minent.
Various foetal malformations (anophthalmia and

microphthalmia, cleft palate, doubling and other
abnormal growth of organs, hydrocephalus).
Embryonic deaths or premature births, still-
births or weakly new-born (cattle, pig, sheep).
Retention of placenta (cattle).
Lactation disorders (mammals).

Reduced hatchability (poultry).
Other symptoms and Low vitamin A content of blood plasma and liver;
diagnostic tests Reduced heat tolerance (cattle);
Test for night-blindness

Examination of the fundus (cattle).
Test for cerebrospinal fluid pressure (cattle, calf).
Human Requirements
The human daily requirement is a minimum of 20-30 lU vitamin A per

kg body weight. However, this is only sufficient to prevent the appearance
deficiency symptoms. About two to three times this quantity is required to
assure an optimal supply. It may be generally assumed that the optimal
requirement of an adult is about 5
,000 lU per day.
:
Provitamin A
Provitamins in general are compounds which are transformed into
vitamins in the body. Provitamins A are among the most widely spread
plant pigments. They occur in fruits (rose-hips, paprika, pumpkins,
apricots, oranges) and vegetables and lettuce). They
(carrots, spinach, kale
are also present in kidney, liver, spleen, milk, butter and cream cheese.
The most important provitamin A is /3-carotene; the carotenoids
derived from it, such as /3-apo-8'-carotenal and the ethyl ester of /S-apo-8'-
carotenoic acid are also important.
Physico-chemical Properties
Structural Formulae
CH3
CH3 CH3 CH
CH3 CH3
/3-Carotene
CH, CH3 CH
CHa
Apocarotenal, R^^CHO; Apocarotenoic Ester, R=COOC2H5
Empirical formulae /3-Carotene C^nHc

'40-^ -^56
Apocarotenal C,nH.nO
^.30x140
Apocarotenoic Ester: C32H44O
Molecular weight ^-Carotene: 5 3^-9

Apocarotenal: 416.6
Apocarotenoic Ester: 460.7
6o Provitamin A
Description /3-Carotene:Reddish-brown to deep violet crys-

powder.
talline
Apocarotenal: Deep violet crystalline powder.

Apocarotenoic Ester Rust red crystalline powder.
:
Solubility ^-Carotene: Insoluble in water, glycerol very

sparingly soluble in alcohol, fats and oils (0.05-
0.08 °o); sparingly soluble in ether, acetone,
slightly soluble in chloroform and benzene.
Apocarotenal and Apocarotenoic Ester Insoluble :
in water, and glycerol, sparingly soluble in alco-

hols, fats and oils {ca. 0.7 °o), slightly soluble in
ether and acetone; readily soluble in chloroform
and benzene.
Melting point /3-Carotene: i76-i82°C

Apocarotenal: 1 36-140 °C
Apocarotenoic Ester : i34-i38°C
Absorption spectrum The three compounds have characteristic absorp-

tion spectra; their solutions in cyclohexane exhibit
the following maxima:
/5-Carotene: about 456 and 484 nm.
Apocarotenal: about 461 and 488 nm.
Apocarotenoic Ester: about 449 and 475 nm.
Stability Like vitamin A, the carotenoids are sensitive to

light, oxygen and acids. They must therefore be
stored in sealed opaque containers from which
the air has been displaced by an inert gas (e.g.
nitrogen).
Methods of Determination
The amounts of /3-carotene in simple mixtures can be determined spectro-

photometrically in the visible light region. In complex, natural mixtures
(foods and feedstuffs) /5-carotene is often accompanied by other carotenoids.
These usually have Little or no provitamin A activity, but frequently show
The latter can only be deter-
light absorption similar to that of /3-carotene.
mined removal of other carotenoids. Procedures suitable for this are
after
absorption and partition chromatography, using column, paper or thin-
layer techniques.
Provitamin A
An "International Unit" of provitamin A is the activity of 0.6 meg of

purest crystalline all-trans- /S-carotene. There is no fixed relationship between
lUs of provitamin A and vitamin A because the vitamin A activity of an
lU of provitamin A varies according to the experimental conditions.
Under the most favourable conditions i lU of provitamin A has an activity
of I lU of vitamin A, but this relationship is rarely found in practice.
According to a proposal of lUPAC (International Union for Pure and
Applied Chemistry) i meg /3-carotene can be equated with i lU of vitamin A
in human foods on the other hand, the World Health Organisation prefers
;
to equate 1.8 meg /3-carotene with i lU of vitamin A. In livestock feeds

the relationship is very variable and somewhat less favourable.
^-Carotene is the precursor of vitamin A. Two possible routes for its
conversion to vitamin A in the intestinal wall have been considered : first,
the central fission of the ;S-carotene molecule, theoretically resulting in two

molecules of vitamin A; and, secondly, the stepwise degradation of the
provitamin at one end of the molecule through the intermediate stages of
/3-apo-8'-carotenal (C,,,) and its corresponding acid. The latter route is
supported by evidence of the natural occurrence of these intermediate

carotenoids and of the transformation vield of vitamin A from ^-carotene
(see structural formulae above).
^-Carotene is markedly fat-soluble and is poorly absorbed. Absorption
is improved by the use of emulsifying agents.
Recent research suggests that ^-carotene has a special function in the
reproductive cycle of the bovine species, which function cannot be per-
formed by vitamin A. The ovaries contain high concentrations of )3-carotene
during the luteal phase and it has been postulated that /3-carotene is involved
in the synthesis of progesterone.
Moreover, in cows on /?-carotene-free diets, "silent" heat was frequently
observed as well as delayed ovulation, follicular cysts, delayed and reduced
formation of the corpora lutea and decreased progesterone level in the serum.
These conditions could be corrected by the addition of /3-carotene to the
ration.
Vitamin D
The antirachitic vitamin D occurs in several forms; the two most
important are vitamin D,, ergocalciferol, formerly known as calciferol,
and vitamin D„ cholecalciferol. These compounds are formed by ultra-

violet irradiation of the sterols ergosterol and 7-dehvdrocholesterol
respectively (provitamins D).
Vitamin D, is found in small quantities in fish-liver oils and some
sponges. Vitamin D, more widely distributed
is in nature, and found in
is
relatively large amounts in fish-liver oils, and fatty tissues, and smaller
amounts are present in hen's eggs, milk, butter and cream cheese.
In addition to the crystalline compounds ergocalciferol and cholecal-
ciferol, the commercial forms of vitamin D also include oily solutions and
stabilised powders.
Structural formulae
Vitamin D, CH3 CH3
CH3 CH3
Vitamin D,
Empirical formulae Vitamin D2 C28H44O

:
Vitamin D, C27H44O
:
Molecular weight Vitamin D,: 396.7

Vitamin D3: 384.6
Vitamin D 63
Description White to yellowish crystalline powders
Melting point Vitamin D,: ii3-ii8°C

Vitamin D,: 82-88 °C
Specific rotation Vitamin D,: [0]^= +102.5 ° to + i07-5

(C=4 in abs. ethanol)
Vitamin D,: [a]^j^= — 105 ° to -^112"
(C=o.5 in abs. ethanol)
Absorption spectrum Vitamins D^ and D, exhibit an absorption maxi-

mum at 265 nm in alcoholic solution.
StabiUty Vitamins D, and D, are destroyed relatively

rapidly by light, oxygen and acids. They must
therefore be stored in opaque, hermetically-sealed

containers from which the air is displaced by an
inert gas, such as nitrogen. The crystalline com-
pounds are relatively stable to heat, but tend to be
isomerised in oily solution.
Methods of Determination in Simple Mixtures
1. Spectrophotor?ietry of the intact structure
The U.V. light absorption property may be used for the assay of pure
preparations free of irrelevant absorption. The U.V. -spectra do not dis-
tinguish between vitamin D, and D,, whereas I. R. -spectra do so.
2. Spectrophotometry after reaction to coloured products

The vitamin Dj and D, give a yellow-orange colour with antimony
trichloride. This colour reaction forms the basis of the U.S. P. XVIII
method for vitamin D. Less widely used reagents are: activated glycerol
dichlorohvdrin, sulfuric acid, trifluoroacetic acid in the presence of hydro-
quinone, furfural and sulfuric acid, stannous chloride and iodine-ethylene
dichloride.These colour reactions are subject to interferences from many
sources and even when combined with chromatographic purification
procedures, the method should be limited to the assay of high potency
substrates such as pharmaceutical preparations and fortified foods and feed
components. Combinations of column and thin-laver chromatographic
purification steps with the antimony trichloride reaction have been success-
fully used.
64 Vitamin D
;?. Gas- liquid chromatography

GLC provides an elegant means of combining qualitative and quantitative
assays. As the vitamins D, and D, as well as their isomerised forms -
silylated or not - are separated, each member of the pair mav be used as
internal standard for the other. However, the GLC methods must still be
preceded by a thorough clean-up procedure when used for composite
substrates.
Biological samples, such as foods, feeds and tissues, contain vitamin D

in very low concentrations and can usually not be assayed by chemical
methods. These substrates will have to be subjected to a biological assav;
as a rule lipid-soluble extracts of the samples are fed to test animals. The
assays measure the alleviation (curative test) or the development (pro-
phylactic test) of vitamin D deficiency in terms of the degree of rickets
produced.
The line test of U.S. P. XV and National Formulary XIII uses stained
longitudinal sections of the distal end of radius bones to evaluate calcifica-
tion. The latter may also be estimated by X-ray radiography or mineraliza-
tion of the bone. The test animal is the rat; however, if the vitamin D
activity of a sample intended for poultry nutrition is to be determined, the
test animal must be the chick, because birds, unlike mammals, only utilize
vitamin D,.
The Internationl Unit of vitamin D was adopted by the WHO as the

standard of activity. It was defined as the biological activity of i mg of an
oily solution of vitamin D, containing 0,025 nicg pure crystallised vitamin D^.
It had been proposed in 1972 to discontinue the distribution of this
standard preparation and to use instead pure crystalline vitamin as the D

standard.
In the United States two further standard preparations are issued by the
U.S. P. Reference Standards Committee (U.S. P. Reference Standards,
46 Park Avenue, York, N.Y. 10016), namely vitamin D capsules for
New
the AOAC-determination in rats, and vitamin D in oilv solution for the
AOAC-determination in chicks.
The U.S. P. Unit corresponds with the International Unit.
In the absence of any of these standards, pure crystalline vitamin D2
or D3 can be used, the activity of 0,025 rncg of either of these compounds
being equated with an International Unit. If preparations of unknown
:
Vitamin D 65
vitamin D content are to be compared with standards, the procedures

described under "Methods of Determination" are used.
Units no longer in use
I M.R.C.-Unit (Medical Research Council) = ca. i lU

I Clinical Unit = ca. 12-17 lU
I International Chick Unit = ca. i lU
I Biological Unit = ca. 0.125 lU
I Protective Unit = ca. 0.125 lU
I Coward Unit = ca. i lU
I Laquer Unit = ca. 0.14 lU
I Poulson Unit = ca. 0.2 lU
I Steenbock Unit = ca. 3 lU
The incorporation of calcium and phosphorus into the bone matrix is

known to be dependent on an adequate supply of vitamin D, which stimu-
lates the absorption of calcium and possibly of phosphorus from the in-
testinal lumen and also increases significantly the re-absorption of calcium

and phosphorus in the renal tubules. Vitamin D thus helps to ensure that
the body has sufficient calcium and phosphorus in the blood for the normal
calcification of bones. Lack of vitamin D, therefore, leads to poor bone
calcification, so that the bones remain soft and liable to bend under load.
These physiological functions are not carried out by the vitamin D
molecule itself. Vitamin D is converted in the body into active forms which
undertake the physiological functions. According to the results of recent
research, the main functional form appears to be the 1,25-dihydroxy-
cholecalciferol formed from vitamin D3 (cholecalciferol) in two enzymatic
hydroxylation stages. 25-hydroxy-cholecalciferol is first formed in the liver
and is then converted into the 1,25-dihydroxy compound in the kidneys.
In addition, other hydroxylated derivatives of cholecalciferol have been
detected in the body. The physiological significance of these vitamin D3
metabolites is, however, still not clear.
General Deficiency Symptoms
Vitamin D deficiency, combined with unbalanced calcium and phos-

phorus uptake, leads to rickets in the young, and to osteomalacia in the
adult.
Rickets is characterised by reduced deposition of calcium in the growing
bone and osteomalacia by a lossof calcium from the fully developed bone.
;
66 Vitamin D
"Ricketty rosary"
(swellings on the
ribs), a charac-
teristic symptom
of rickets (chicken).
General condition Poor appetite, stunted growth and weight loss;

dejected appearance (cattle, pig, chicken).
Nervous system and Increased irritability and incidence of convulsions

muscles due to tetany (piglet).
Bones Stiff, painful gait, immobility, painless hard

swelHngs on limb joints and ribs ("rosary"), bone
distortion in limbs, breast-bone (poultry) and
backbone, increased bone fragility, soft pUable
bones (pets and farm livestock).
Fertility Decrease in egg production, thin-sheUed eggs,

reduced hatchabiUty (chicken);
Birth of weak, dead or malformed young (cattle,
sheep)
Embryonic malformations (3rd to 7th day,
chicken).
Vitamin D 67
Very severe rickets

in a pig.
Human Requirements
The vitamin D requirement of man is naturally liigh during the period of

groMt'th. Duringthis time 400-500 lU vitamin D per day are sufficient to
ensure optimal supply. Adults who spend enough time out of doors in
sunlight should obtain sufficient vitamin D from natural sources and from
the conversion of provitamin D.
Additional vitamin D appears to be indicated for those who work at
night or underground and for the elderly.

The vitamin D requirement also increases during pregnancy and lacta-
tion to 500-1000 lU per day.

:
Vitamin E
Naturally occurring vitamin E comprises a series of compounds called

tocopherols. Among the richest sources of vitamin E are cereal germs and
most oilseeds. Vitamin E is also found in leafy vegetables (lettuce, spinach,
cabbage, leek), in animal organs (pituitary, adrenals, pancreas, spleen) and
also in milk, butter and abdominal fat.
The most important representative of the vitamin E group is a-toco-

pherol, which is marketed chiefly as the acetate in the form of an oil or a
stabilised powder.
Structural formulae
CH3
CHs
dl-a-Tocopherol: R=H; dl-a-Tocopheryl acetate : R=CH3-CO
Empirical formula Tocopherol : C2gH5o02

Acetate: C31H52O3
Molecular weight Tocopherol: 430.7

Acetate: 472.8
Description Bright yellow viscous oils.
Solubility Insoluble in water, readily soluble in alcohol,

organic solvents and vegetable oils.
Refractive index Tocopherol: 1.5 04-1. 5 07

Acetate: 1.49 5-1. 498
Absorption spectrum Tocopherol : In alcohol solution

Max. at 292 nm
Min. at 2 5 5 nm
Acetate In alcohol solution
Max. at 284-285 nm
Min. at 254 nm
Vitamin E 69
Stability dl-a-Tocopheryl acetate is relatively stable to-

wards atmospheric oxygen; it is hydrolysed by
moisture in the presence of alkalies or strong acids
to free tocopherol, which is rapidlv oxidised bv
air with darkening of colour.
Methods of Determination in Media of Simple Composition
1. U.V. region
Spectrophotometry in the
The absorption spectrum of free or esterified tocopherol mav be used
for quantitative determination in pure solutions. However, as its maximum
is situated at rather short wave-lengths, background absorption often
becomes it is preferable to measure the difference
a problem. In these cases,
in absorbance between tocopherol and tocopheryl quinone after oxido-
reduction which is the basis of most tocopherol assays.
2. Spectrophotometrj in the visible region of the spectrum

Free a-tocopherol isoxidized by nitric acid to a reddish reaction product
called tocopherol red. With 90 °o sulfuric acid, a yellow colour is obtained.
The ester bond of a-tocopheryl acetate may be split in the presence of
hydroxylamine yielding an equimolar amount of hydroxamic acid which
may be quantified as the red Fe™ complex. These colorimetric reactions
are subject to interference from various sources and are therefore only
suitable for high potency preparations of known composition.
In those colorimetric assays which are based on the oxidoreductive
behaviour of free tocopherol, the chromogen is obtained in a coupled
reaction. The best known and still extensively used technique of this t}ê
isthe Emmerie-Engel reaction. One molecule of free a-tocopherol reduces
two Fe™ ions to Feî ions. The latter form a red complex with 2,2'-dipyridyl
whose concentration is determined colorimetricaUy at 520 nm. A variety
of chelating agents containing the ferroin chromogen mav be used, such
as, for instance, o-phenanthrolin or bathophenanthrolin (4,7-diphenyl-i,io-
phenanthroUn. Use of the latter seems gradually to replace the older 2,2'-
dipyridyl due to its better colour yield.

Alternative means of using the reductive properties of tocopherol for
colorimetry are the production of molybdenum blue with phosphomolybdic
acid and the discoloration of the stable radical i,i-diphenyl-2-picryl-
hydrazyl (DPPH).
). Oxidimetric methods with non-photometric detection systems

a-Tocopherol is oxidised by eerie sulfate to a-tocopheryl quinone. The
reaction may be followed by indicator or amperometric titration.
yo Vitamin E
The reduction step from tocopheryl quinone to tocopherol is suitable

for polarographic assay, preformed quinone being measured direct and total
tocopherol after preliminary oxidation to the quinone.
The voltametric determination of tocopherol by use of a carbon paste
electrode has also been described. The oxidation of tocopherols with Fe™
ions, which is usually the basis of photometric assays, may also be quantified
by back titration of the Fe^^ ion with dichlorophenol-indophenol in the
presence of metaphosphoric acid or of the excess Fe^ ion using potentio-
metry with sodium ethylene diamine tetra acetate.
./. Flmrometry
Solutions of a-tocopherol fluorescence in the U.V. region. Esterified
tocopherol and the quinone do not show any measurable fluorescence. A
fluorescent derivative of tocopherol is obtained by reacting tocopherol
with nitric acid to tocopherol red and condensing the latter product with
ortho-phenylene diamine. The resulting phenazine shows a yellow-green
fluorescence with excitation in the near U.V.
The assay of tocopherol in foods, feeds and medium or low potency

pharmaceutical mixtures is mainly a clean-up and separation problem, as
the above-mentioned reactions are either not specific or suff"er from matrix
interference. The older purification techniques using molecular distillation,
catalytic hvdrogenation and estimation of non-a-tocopherols by nitrosation
have more or less been abandoned. Usually a lipid soluble extract of the
sample is prepared and subjected to a chromatographic separation either
direct or after alkaline saponification or after low temperature crystallisation
of the bulk of the lipids.
Protection of tocopherol from oxidation during the various steps of

the analytical procedure is absolutely necessary. Conditions which catalyse
the oxidation of tocopherol by traces of dissolved oxygen are light, heat,
alkaline pH, and metals. It is not possible to mention all the individual
chromatographic methods for tocopherol assay as there are too many. In
general it may be stated that open column chromatography is still used, but
mainly as part may be freed of vitamin A,
of the clean-up process. Extracts
carotenoids, sterols, by passage through Fuller's earth, silicagel,
etc.
alumina, zinc carbonate, magnesium oxide and others. Columns of florex

treated with eerie sulfate are used to separate the tocopherol acetate added
as fortification from the tocopherol naturally present, and from other
reducing impurities which are oxidised, leaving the ester unchanged.
1
Vitamin E 7
Liquid-gel partition chromatography in open columns has also been

described.
Separations with high resolution are obtained using two-dimensional
chromatography on zinc carbonate impregnated paper, various thin-layer
chromatography systems, which are presently the most widely used tech-
nique, and gas-liquid chromatography. The latter, very convenient,
techniques will probably be chosen as official methods of analysis in the near
future. The tocopherol is separated as such or is transformed to an ester,
like the acetate and trifluoroacetate, or to the trimethyl silyl ether before
GLC assay.
High pressure liquid chromatography will certainly become the method
of choice for tocopherol analysis as soon as the equipment is as widely
available as GLC at the present.
The chosen International Unit is mg of synthetic dl-a-

the activity of i
tocopheryl acetate. Preparations of unknown vitamin E activity are tested

especially in the so-called resorption sterility test or haemolysis test in the
rat. The rat resorption sterility test is based on the phenomenon of resorp-
tion of the foetus in a pregnant female rat, under vitamin E deficiency,
between the eighth and sixteenth days of pregnancy. The haemolysis test
depends on the fact that, in the presence of dialuric acid, the blood of
vitamin E-deficient animals haemolyses more rapidly than that of normal
animals. Direct comparison of the preparation to be analysed with the
standard must be strictly maintained; thus, from a comparison of so-called
1-a-tocopherol with dl-a-tocopheryl acetate, conclusions cannot be drawn
as to the activity of d-a-tocopherol, since synergistic effects may be expected
from simultaneous administration of different tocopherols.
One Unit no longer used is the Median Fertility Dose (MFD) which is
equivalent to i lU of vitamin E.
The following relationships hold for some important members of the

vitamin E group:
I lU of vitamin E is equivalent to
I mg dl-a-Tocopheryl acetate
0.909 mg dl-a-Tocopherol
0.735 mg d-a-Tocopheryl acetate
0.671 mg d-a-Tocopherol
1.75 mg d- ^-Tocopherol
7 mg d-y-Tocopherol
-jz Vitamin E
The great importance of the physiological action of vitamin E shows

itself in the manifold deficiency symptoms that appear in the absence of
the vitamin. As a fat-soluble intracellular antioxidant vitamin E is involved,
above all, by inhibiting the formation
in stabilising unsaturated fatty acids
of toxic lipoperoxides. In the body vitamin E also protects the oxygen-
sensitive vitamin A from oxidative destruction, thus improving the vit-
amin A supply.
The damage of blood vessels and changes in capillary permeability that
occur in vitamin E deficiency are probably also related to these antioxidant
properties. Furthermore, vitamin E is an important protective factor for
ervthrocvte membranes; it increases their resistance to haemolvtic agents
such as hydrogen peroxide and dialuric acid.
In addition to its activity as a physiological antioxidant vitamin E exerts

a range of other specific vitamin functions, but their mechanisms of action
are still far from clear. Special mention should be made of the action of
vitamin E in maintaining testicular function. In female animals vitamin E
deficiency leads to foetal resorption (resorption steriHty). Further, it
prevents muscular degeneration and liver necrosis.

According to more recent studies vitamin E influences the metabolism
of nucleic acids and polyunsaturated fatty acids. It also has a regulatory
action on the pituitary-midbrain system. Vitamin E promotes the production
of thyrotropic and adrenocorticotropic hormones and the gonadotropins,
whereas in vitamin E deficiency the hormone content of the pituitary falls.
In these cases, too, the mechanism of action of vitamin and the possible E
connections between the metabolic disturbances and the outward clinical
symptoms have not yet been determined.
Vitamin E is present in practically all body tissues; uterus, testes,
adrenals and pituitary have particularly high vitamin E levels, compared
with other organs, which agrees well with the specific physiological func-
tions of the vitamin in these organs. In the liver, vitamin E is localised
mainly in the metabolically active cellular particles, the mitochondria and
microsomes.
Investigation of experimental vitamin E deficiency in laboratory animals

has shown whole range of the most varied symptoms may be found,
that a
depending on external and internal factors such as composition of diet or
animal species. These symptoms have little in common; usually, however,
the condition is of a chronic nature and the deficiency disease only leads
to death of the animals in the rarest cases.
;
Vitamin E 73
This may account for the fact that in man little or no clinical vitamin E
deficiency symptoms have been established with certainty.
Deficiency states are, however, important in livestock since they fre-
quently appear quite suddenly and present very varied disease pictures.
The effect of a chronically deficient supply of vitamin E is accentuated if,
in addition, there is a deficiency of protein (appearance of E-deficiency
anaemias) or of selenium (occurrence of liver necrosis) or if large amounts of
fat containing unsaturated fatty acids (e.g. fish-liver oil) are also adminis-
tered.
General condition Growth disturbance (irregular)
Nervous system Encephalomalacia (poultry)
Muscular system Degeneration of heart and skeletal muscle (all
domestic animals, livestock)

Sudden heart failure (pig, calf, lamb).
Fatty tissues Increased oxidisabiUty of body fat ("Yellow fat

disease", Steatitis). (mink, fox, cat, pig, poultry).
Poor muscles and

lacic of vitality due
to vitamin E
deficiency in a
new-born lamb.
; ; :
74 Vitamin E
" Yellow fat

disease". Mink
Blood and Increased susceptibility to haemolysis of erythro-

cardiovascular system cytes (rat, pig);
Anaemia (ape);
Exudative diathesis (poultry).
Liver Hepatosis Diaetetica (pig).
Reproductive Testicular degeneration (piglet, calf, poultry,

svstem dog);
Foetal deaths (pig)
Debility and muscular degeneration of new-born
(piglet, lamb, calf).
Further symptoms, Increased rate of haemolysis under oxidative

diagnostic tests treatment
Limited specific tests
Increased glutamic-oxalacetic acid transaminase
and aldolase activity in blood plasma (pig, cattle,
sheep).
Vitamin E 75
Clinical appearance
of encephaloma-
lacia: "Crazy chick
disease".
Encephalomalacia
due to vitamin E
deficiency in the
chick: Hyper-
plasias and haemor-
rhages of the cere-
bellum. Healthy
control animal in
upper part of
picture.
76 Vitamin E
Human Requirements
The daily requirement of man for vitamin E is estimated at 30 lU.

In pregnancy and for the elderly about twice as much is necessary to achieve
an optimal supply. The daily requirement of an infant is 5-10 lU. The
vitamin E requirement of the body increases with increased dietary ad-
ministration of unsaturated fatty acids (linoleic and other fatty acids).
Vitamin K -F^M^luChu
The anti-haemorrhagic vitamin K, which has an important role in

regulating blood coagulation, occurs in anumber of forms; chemically,
these are derivatives of 2-methyl-i,4-naphthoquinone bearing a side-chain
in the 3-position. In the vitamins Kj, one of the main groups, the side-chain
has only one double bond, whereas in the other group, K2, double bonds
recur regularly in the side-chain.
Vitamin K; is present in green plants (stinging nettles), green vegetables
(cabbages. spinach\ potatoe s, fruits ( to matoes, strawberries, rose-hips)
and also in liver-oils. V itamin K, has been found in animal and microbial
materials.
The most important member of the vitamin K group is the compound
in the K, series with twenty carbon atoms in the side-chain, generally
known as vitamin K,; the most usual synonyms are Phytonadione (U.S. P.)
and Phytomenadione (B.P.).
The essential structural feature of all K- vitamins is that of 2-methyl-i,4-
naphthoquinone ; this compound, also known as menadione, and sometimes
as vitamin K,, also has vitamin K activity, although this is qualitatively
different in that it is not an antagonist of the anti-coagulant drugs such as
dicumarol and warfarin. It forms a similarly active water-soluble sodium
bisulphite compound (water-soluble K,). A further active and soluble
derivative is the sodium salt of the corresponding naphthohvdroquinone
diphosphate.
Phjsico-chemical Properties (Vitamin KJ
Structural formula
Empirical formula
Molecular weight 450.7
Description Golden yellow viscous oil
Solubility Insoluble in water, sparingly soluble in alcohol,

readily soluble in ether, chloroform, fats and oils.
Absorption spectrum Vitamin Kj in cvclohexane shows maxima at 243,

249, 261 and 270 nm and minima at 2 54and 285 nm.
78 Vitamin K
Refractive index [n]Q = i. 525-1. 528
Stability Vitamin Kj is sl owly degraded by atmospheric

oxygen but fairly rapidly destroyed by light. It is
K I htZ-^JiîA><J^ ^ ^HJ relatively stable to heat, but decomposed by alkalis.
Menadione Sodium Bisulphite (water-soluble vitamin K3)
Structural formula
Empirical formula CuHgOj-NaHSOj-xHzO
Vitamin K^: In pure solutions, vitamin K, may be assayed by U.V.

photometry. Additonal qualitative information and means of distinguishing
between vitamin Kj and related structures are obtained from the determina-
tion of the isosbestic points in the spectrum upon reduction of the quinone
moiety.
Colorimetric determinations, also in highly purified extracts, are made
using either the Irreverre-SulUvan test which yields a blue colour upon
reaction of vitamin K, with diethyl dithiocarbamate and sodium ethylate
in ethanoUc solution or the Schilling-Dam test which yields a yellow-
orange colour when the vitamin is treated with xanthane h\dride (5-imino-
3-thion-i,2,4-dithiazolidine) in ethanolic potassium hydroxide.
Other accepted techniques are the oxidimetric assay after catalytic
reduction to the hydroquinone and the polarographic determination.
Vitamin Kj shows a phosphorescence spectrum which is of potential
use for analytical purposes, no fluorescence is detectable with the intact
compound.
The assay in biological material requires the separation of the vitamin
from accompanying substances of related chemical structure and from
comparatively large amounts of lipid soluble co-extractives. The instability
of vitamin Kj precluding saponification, the clean-up is usually made by
freezing out, by counter-current distribution or by direct open column
Vitamin K 79
chromatography. The final determination is performed by chromatography
on alumina impregnated paper, elution and U.V. photometry, by thin-layer

chromatography with U.V. densitometric measurement or by gas chromato-
graphy using flame ionization detection.
Because of the low concentrations of yitamin K, encountered in bio-
logical samples and the lability of the compound, the animal test has not
been superseded. The biological test measures the coagulability of the blood
of chicks (or rats) and may be carried out prophylactically or curatiyely, the
latter being usually preferred. The principle of the curative test is determina-
tion of coagulation time of the blood of yitamin K-deficient animals before
and after ingestion of graded amounts of an unknown sample versus pure
yitamin K substance.
Menadione: Pure solutions of menadione or menadione sodium bisulfite

may be assayed by U.V. photometry. As the spectra of the two forms differ,
the pH and sodium bisulfite concentration of the measuring solution must

be controlled.
Colorimetric assays of menadione are based on the absorbance charac-
teristics of the 2,4-dinitro-phenyl hydrazone in ethanolic ammonia (blue
colour), the reaction product with ethyl cyanoacetate in ethanolic ammonia
(Craven's test, blue colour) and the reaction product with hydrochloric
acid and zinc chloride (blue-red colour). The colour reaction of menadione
with cysteine in alkaline solution is now seldom used.
The oxidimetric assays comprise a reduction of menadione to the hydro-
quinone and reaction of the latter with an oxidant such as eerie sulfate or
dichlorophenol-indophenol. End-point determination is usually by poten-
tiometry. A microcoulometric assay has also been described. Several
techniques using polarography are available, which follow the reduction
of menadione involving two electrons to produce the corresponding hydro-
quinone.
Menadione, like vitamin Kj, shows a phosphorescence spectrum, which
could possibly be used for assay purposes. A condensation product between
menadione or its hydroquinone and ortho-phenylene diamine shows
fluorescence in the visible part of the spectrum. The assay has, however,
not been used extensively.
In complex mixtures, a chromatographic separation must precede the
assay. Several techniques using paper, thin-layerand gas liquid chromato-
graphy have been described. The latter technique is very convenient, as it
combines good separation efficiency with quantitative detection in the flame
ionization detector.
Mixtures containing low concentrations of menadione can also be
assayed by the biological test. Here, however, the curative test in chicks
should be used, since menadione does not normalise the blood clotting time
of the dicoumarol-poisoned rabbit.
Vitamin K
No international standard for the biological activity of vitamin K has

been defined. Since there is a fundamental qualitative difference in activity
between menadione or its derivatives and vitamin K, and its analogues,
either pure menadione or vitamin Kj must be taken as a standard, depending
on which is in question.
Both of the biological tests mentioned above may be considered for
the biological standardisation of preparations with vitamin K activity.
Units that are now obsolete are:
1. Almquist-Unit (activity equivalent to ca. i6 meg vitamin Kj or
4.2 meg menadione);
2. Dam-Glavind-Unit (D.G.U.) (activity equivalent to ca. 0.083 "^^g
vitamin K, or 0.04 meg menadione).
Vitamin K is indispensable for maintaining the function nf the blood-

coagula tion system. It participates - bv a mechanism still not fully under-
stood - in the formation of prothrombin (Factor and of the clotting II)
factors VII, IX and X, so that a vitamin K deficiency markedly reduces

the normal speed of blood-clotting and thus causes haemorrhagic problems.
These processes are depicted below in a highly simplified scheme. This
shows that the concerted action of the different factors and the activated
thromboplastin transform the precursor prothrombin into the clotting
Vitamin K and Blood Coagulation

Factor V Factor XI
. \ Factor VlF Faaor XII
Factor VIII Platelet
Formation factor
dependent <yi:i Factor IX

on Calcium
vitamin K Factor .X ions
Active plasma and tissue

/y^. thromboplastin
e^;2^Mu^
\k \l/
Prothrombin hrombin
/W I Fibrinogen
i > " Kbrin
Vitamin K
Anaemic appearance
of fowl resulting
from (experimental)
vitamin K defi-
ciency.
enzyme thrombin^ which, in turn , initiates the actual clotting phase by

converting the soluble fibrinogen to the insoluble fibrin.
Menadione (2-methyl-i,4-naphthoquinone; "vitamin K3") can be con-
verted in the body to vitamin K
(probably a vitamin Kj-type of compound
with twenty carbon atoms in the side-chain). The linkage of menadione
with an isoprenoid side-chain as in the naturally occurring vitamin K
appears to be necessary for the biological activity of menadione.
Vitamin K
deficiency can result not only from inadequate dietary supply
but also from disorders of absorption. Deficiency symptoms can also appear
in animals if adequate synthesis by the intestinal flora cannot take place, for
example in the administration of antibiotics or sulphonamides or through
the effect of anti-coagulants, such as dicoumarol which may be present in
spoiled sweet-clover and in certain rodenticides (dicoumarin or indanedione
derivatives).
Vitamin K deficiency leads uniformly in man and all investigated animals
to a fall in the prothro mbin content of the blood, eliciting a
haemorrhagic
tendency and haemorrhages in the most varied tissues and organs (the sub-
cutaneous tissue, muscles, brain, gastro-intestinal tract, abdominal cavity,
uro-genital organs etc.) and which can, especially, give rise to complications
in neonatal and premature birth cases in humans. All other symptoms must
be regarded as consequences of this phenomenon.
82 Vitamin K
Haemorrhages
in the subcutaneous
tissue.
Blood and Numerous haemorrhages in the subcutaneous and

circulation other tissues and organs;
Anaemia.
Other symptoms and Increase in blood-clotting-time,

diagnostic tests
Human Requirements
Since the m^gygg^ r\iri <c .jaîfficientlv developed in the first days
after birth to provide, the vitamin K requirement of the
and because
infant,
of the low vitamin K content of the mother's milk, the prothrombin level
is very low in the new-born. A daily dose of 1-2 mg vitamin K for new-born
infants, or 2-5 mg daily to the mother before confinement, is therefore

recommended. The daily requirement of adults is estimated at about i mg.
H i
Vitamin C
It known for centuries that scurvy, one of the oldest diseases

has been
known man, could be treated with fruits and vegetables or their juices,
to
but it was only some forty years ago that the anti-scorbutic vitamin C was
identified as L-ascorbic acid, the most important of the various compounds
that possess vitamin C activity.
Ascorbic acid occurs in all living tissues as an important redox-compound

of cell metabolism. Important sources of vitamin C ar e fresh fru its (citrus,
black curran ts, rose-hips , sea buck-thor n, paprika, tomatoes)
and vege-
tables (cabbage, potatoes, lettuce)Animal organs showing a notable content
.
of ascorbic acid are the adrenals, liver and pituitary.

The ene-diol group at carbon atoms 2 and 3 of L-ascorbic acid is readily
oxidised to a diketo group. The resultant dehydro-L-ascorbic acid is
equally active as vitamin C, and forms a redox system with the reduced
compound. In addition to L-ascorbic acid its sodium salt (sodium ascorbate)
is frequently used in practice.
Structural formula
O^C-
C — OH
C — OH
II
—C
H *
HO — C —
I
CH2OH
Empirical formula Ascorbic acid: C^HgOg

Sodium salt: C6H706Na
Molecular weight Ascorbic acid: 176.1

Sodium salt: 198.
Description White to yellow tinged crystalline powder.
SolubiUty Ascorbic acid: Readily soluble in water (ca. 30 g/
100 ml), slightly in alcohol, sparingly soluble in

glycerol, insoluble in ether and chloroform.
84 Vitamin C
Solubititv Sodium salt: Very soluble in water (ca. 90

g/
100 ml), practically insoluble in alcohol, ether and
chloroform.
Melting point Ascorbic acid: ca. 190 "C (decomposition)

Sodium salt: Decomposition, without sharp
melting point, at ca. 220 °C.
Absorption spectrum In U.V. light ascorbic acid in strongly acid

solution shows an absorption maximum at ca.
245 nm, which shifts at neutrality to 365 nm and

at pH 14 to ca. 300 nm.
Specific rotation Ascorbic acid: [a]^=-zz^ to -23°

(c=2 in water)
Sodium salt: [a]^j5= — 103 '^ —106°
(c = 5 in water)
Stability Crystalline ascorbic acid is relatively stable in air,

if moisture is completely absent, while the sodium
salt tends to turn yellow. Aqueous solutions are
attacked by atmospheric oxygen and other oxidis-
ing agents; the first-formed dehydroascorbic acid
is further and irreversibly oxidised, alkalies and
traces of heavy metal ions (e.g. copper ions)
acting as catalysts.
Most of the assay methods for vitamin C are based on the reductive
properties of the carbonyl-ene-diol group of ascorbic acid, which is oxidised
yielding dehydro-ascorbic acid. Suitable oxidimetric reagents are 2,6-
dichlorphenol-indophenol (Tillmans reagent), iodine, chloramine-T,

N-bromosuccinimide, sodium i,2-naphtoquinone-4-sulfonate and many
others. The end-point determination is by visual indicator titration, photo-
metry or potentiometry. The assay by polarography is also widely used. The
disadvantage of the oxidimetric methods is their tendency to overestimate
the potency of the sample when foreign substances with redox potentials
similar to that of ascorbic acid are present.
Non-oxidimetric assay procedures are the reaction of ascorbic acid with
diazotised aniline derivatives, e.g. 4-methoxy-4-nitroaniline, yielding the
-
Vitamin C
corresponding half-hydrazides of oxalic acid which are coloured in alkaline

solution and the reactions of dehydroascorbic acid at its dicarbonyl site.
The best known assays of dehydroascorbic acid are the colorimetric

determination of the bis-2,4-dinitro phenyl hydrazone and the fluorometric
determination of the quinoxaline obtained by condensation with ortho-
phenylene diamine. The latter reaction performed with suitable derivatives
of phenylene diamine also permits a photometric evaluation.
More recent methods, which are not yet in widespread use, are the gas-
liquid chromatography of the trimethyl silyl derivative of ascorbic acid
and the electrochemical monitoring of ion-exchange column effluents with
a carbon paste electrode as detector.
Assay procedures applicable to complex mixtures of low potency and
biological material usually combine chromatographic separations with one
of the above-mentioned measuring procedures. The methods which separate
ascorbic acid itself by paper chromatography or paper electrophoresis have
their limitations, mainly because of the lability of the redox system ascorbic
dehydroascorbic acid in aqueous solution and interferences of foreign ions.
Chromatography of the dinitrophenvl hydrazone or other derivatives of
dehydroascorbic acid is well accepted but cumbersome. More recent
trends of vitamin C analysis are the use of non aqueous solvents for ex-
traction, especially from dry materials, such as feedstuffs, and of a new
reducing agent, dimercaptopropanol, in order to inhibit or reverse the
formation of dehydroascorbic acid by catalyzed air oxidation. Ascorbic
acid can be trapped on an anion exchange resin and the non acidic coextrac-
tives washed out. Then, the ascorbic acid is oxidised to dehydroascorbic
acid by p-benzoquinone on the exchanger, whereby it loses its acidic
character and is eluted from the column. This most efficient clean up
procedure allows for practically any final assay technique and even chroma-
tographic investigation of the eluate, for instance to distinguish between
L-ascorbic acid and ervthorbic acid.
The "International Unit" of vitamin C is the activity of 50 meg of pure,

crystalline L-ascorbic acid. The International Unit is, however, not com-
monly used as a measure of vitamin C activity ; this is measured much more
widely in terms of weight units of pure, crystalline ascorbic acid.
Of the few animal species for which L-ascorbic acid is an essential
nutritional factor, only the guinea-pig is used in As criteria for vit-
tests.
amin C action the prevention or cure of scurvy symptoms and the growth
of odontoblasts of incisors can be used.
86 Vitamin C
Vitamin C is essential both for the formation of intercellular substances
of skeletal tissues (connective tissue, bones, cartilage, dentine) and also

for maintenance of the normal function of these tissues. The vitamin also
exerts a stimulant action on the defensive mechanisms of the body (phago-
cytic activity of the leucocytes, reticuloendothelial system, formation of
antibodies).
Since ascorbic acid is readily oxidised to dehydroascorbic acid and this
can be reversed by reduction to ascorbic acid, it is suggested that vitamin C
participates as an oxidation-reduction system in cellular oxidation processes.
The impaired formation of the intercellular substance of skeletal tissues in
vitamin C deficiency is related to disturbance of the hydroxylation of
proline to hydroxyprohne, an important structural component of collagen
fibre.
According to more recent results vitamin C plays an essential role in

the transport of iron ions from the plasma protein transferrin to the organ
(?^ pr otein ferritin which serves in the storage of iron in bone marrow, spleen
and liver. Vitamin C deficiency disrupts this transport of iron between
jlasma and storage organs.
Certain inter-actions appear to exist between L-ascorbic acid and folic
acid since i n vitamin C deficiency an increased requirement for folic acid
is observed.
While man, the primates, the guinea-pig and fish (so far investigated)
are dependent on their food for supphes of vitamin C, most other animal
species can themselves synthesise the vitamin from D-glucose or D-galac-
tose. For these animal species therefore L-ascorbic acid is not strictly a
vitamin.
In man and the other animals which are incapable of endogenous syn-
thesis of L-ascorbic acid a specific enzyme required in the synthetic chain
is missing (L-gulonolactone oxidase).
Vitamin C has a special position among the vitamins inasmuch as only

man and certain laboratory and wild animals depend on a daily dietary
supply of this substance. Severe deficiency symptoms are therefore only
known in man, characterised as scurvy in adults and as MoUer-Barlow
disease in children.
Besides these more pronounced deficiency symptoms there also occur
in humans and in livestock a range of nonspecific symptoms once there is
too little vitamin C available to the body, as the result of either insufficient
supply or inadequate synthesis. Hypov ita minoses_o f this kind are difficult
Vitamin C 87
to recognise, and show themselves in man in the form of weakness, fatigue,
dyspnoea, aching bones, hyperaesthesia and pain in the lingual and buccal
mucosa, follicular hyperkeratoses, swelling and bleeding of gums, and
haemorrhagic diathesis.
Calves and piglets appear to be unable to synthesise adequate amounts
of vitamin C during the first 10 days of life. Further, some fully grown
domestic animals can clearly show a reduced synthetic ability or an increased
requirement for vitamin C, induced by various stress factors (heat, cold,
infections and parasitic diseases, poor or unsatisfactory diet). In poultry

this can lead to deterioration in growth, egg production and quality of egg-
shells.
Scurvy Inhibition of growth, anorexia, loss of ac t ivity,

diffuse bleeding in skin and joints, bone fractures,
loss of fur sheen loss in weight, anaemia, diarrhoea
,
~~ '
(guinea-pig, monkey).
Human Requirements
Unlike most animal species the human body cannot synthesise vitamin C.
In view of the numerous functions of this vitamin in man an adequate
supply is extraordinarily important. The following values can be given for
the daily requirement:
Men 75 mg
Women 70 mg
Pregnant women 100 mg
Lactating women 1 5 o mg
Infants 30 mg
Children 1-3 years 35 mg
Boys 13-15 years 90 mg
Boys 16-19 years 100 mg
Girls 13-19 years 80 mg
The requirement is increased during intensive physical exertion, feverish
illness, hyperthyroid activity, diabetes, after surgical operations and during

high liquid intake.
:
88 Vitamin C
Guinea-pig on
normal diet.
Guinea-pig in
C-hypovitaminosis
prone position,
taking weight off
atrophied leg mus-
cles.
Effect of vitamin C
on ossification and
growth of tibia
(guinea-pig)
C-free diet (I);
Addition of
0.25 mg (II),
I mg (III),
5 mg (IV), per
kg
body weight per
day.
Vitamin Bi
Vitamin Bj is also known as thiamine, from the thiazole and pyrimidine

rings in the structure; the synonym aneurin relates to the antineuritic
properties of the vitamin. Reflecting its important function in carbohydrate

metabolism, vitamin Bj is present in almost all living tissues. It occurs in
the pericarp and germ of cereals, in yeast, vegetable, fruits and potatoes,
also in animal organs (mainly in liver and kidney) as well as in egg-yolk and
milk.
Thiamine is used chiefly in the form of the chloridehydrochloride
(usually designated commercially as thiamine hydrochloride); the mono-
nitrate is also of some importance.
Structural formula
N^^^l CH2 N 1
CH3
CH2 — CH2OH
Hydrochloride: X=C1-, HCl
Monorutrate : X=NO,-
Empirical formula Hydrochloride: Ci^H.ÔN^ClS. HCl

Mononitrate: C,,Hi-04N5S
Molecular weight Hydrochloride: 337.3

Mononitrate: 327.4
Description Hydrochloride: White crystalline powder

Mononitrate: White crystalline powder
Solubility Hydrochloride: Readily soluble in water (ca.
I g/ml), sparingly soluble in alcohol, practically

insoluble in ether, hexane, chloroform, acetone,
benzene.
Mononitrate: Slightly soluble in water (ca.
2.7 g/ 00 ml), sparingly soluble in alcohol chloro-

1
form.
Melting point Hydrochloride: ca. 250°C (decomposition)

Mononitrate: ca. 1
90-200 °C
oo Vitamin B,
Absorption spectrum Thiamine shows a characteristic absorption

spectrum in the region 200-300 nm. The positions
of the maxima and the respective extinctions
depend markedly on the solvents used and the
pH of the solutions. In o.i N hydrochloric acid
solution thiamine shows an absorption maximum
at ca. 245 nm.
Stability In the absence of light and moisture thiamine

salts are relatively stable towards atmospheric
oxygen even when warm. Acid solutions are also
fairly stable; in neutral or alkaline solution,
however, decomposition occurs.
Methods of Determination in Media of Simple Composition
The most widely used assay for vitamin B, is the fluorometric determina-
tion. The method is based on the oxidation of thiamine in alkaline solution
to the strongly fluorescent derivative thiochrome.
There are, however, a great number of other techniques which are
suitable for high potency samples of known composition. For instance, the
U.V. absorption of thiamine itself may be used for spectrophotometry, or
the colour of condensation products of thiamine with a variety of diazotised
aromatic amines such as p-aminoacetophenone, p-aminobenzoate, 6-amino-
thymol and others, for colorimetry.
There are also techniques based on the formation of insoluble precipitates
of vitamin Bj with reagents such as silicotungstic acid, lithium picrolonate,
ammonium reineckate or iodobismuthic acid. The corresponding measure-
ments are performed by back titration of the reagent in excess, by colori-
metry or by gravimetry.
A direct titration with perchloric acid in glacial acetic acid which may
be followed potentiometrically is available and also several methods using
polarography.
Combinations of the fluorometric technique with paper, thin-layer, ion-
exchange chromatography or electrophoresis are used in various assay
procedures.
Usually, a combination of the thiochrome procedure with enrichment

and purification steps is used. The samples (foods, feedstuff"s, etc.) are
autoclaved in acid medium and treated with enzyme preparations to cleave
Vitamin B, 91
the phosphate ester bonds of thiamine phosphate esters. Sometimes, a

reductive splitting of thiamine disulfide bonds is also included in the
procedure. The extract is purified and the thiamine concentrated by chroma-
tography on zeolith (Decalso) or carboxylic kation exchange resins and
submitted to the final assay.
Alternatively, a microbiological assay may be used. Among the organisms

suitable for this are: Phy corny ces blakesleanus, ochromonas danica, neurospora
Another kind of microbiological
crassa, lactohacillus fermenti, kloeckera hrevis.
assay is the yeast fermentation method. It is based on the fact that the rate
of alcohol fermentation bv living yeast can be increased in proportion to
added thiamine.
Enzymatic methods are suitable for the determination of the pyrophos-
phate ester of thiamine (cocarboxylase). For instance, the decarboxylation
of pyruvate by the thiamine pyrophosphate dependent enzyme (a-oxo-acid
carboxylase) to yield acetaldehyde and carbon dioxide may be coupled
with an indicator reaction using the reduction of acetaldehyde with NADH
in the presence of alcohol dehydrogenase. The rate of disappearance of
NADH in the test system is a measure of the thiamine concentration available
for reconstitution of the holoenzyme from the apo-decarboxylase.
The "International Unit" of vitamin Bj is the activity of 3 meg of

thiamine hydrochloride; the U.S. P. Unit is also based on this relationship.
Neither unit, however, is used in practice; the weight unit (mg) of thiamine
hydrochloride is used as a measure of vitamin Bj activity.
The biological activity of unknown preparations can be determined

by the curative growth test or the so-called bradycardia
either test in rats, in
comparison with a standard preparation of vitamin Bj.
Vitamin B,, form of its pyrophosphate ester (cocarboxylase),

in the
participates in thenormal process of carbohydrate metabolism. As co-
enzyme of the pyruvate dehydrogenase and a-ketoglutarate dehydrogenase
complexes vitamin Bj acts in the oxidative decarboxylation of these car-
boxylic acids ;
pyruvate is transformed into active acetate (acetyl-coenzyme
A) and a-ketoglutarate into active succinate (succinyl-coenzyme A). Active
acetate has important roles in cell metabolism (see Pantothenic acid, p. 125).
It feeds into the citric acid cycle and is oxidised to carbon dioxide and water
by the combined action of the cycle and the respiratory chain (see Nicotinic
acid p. 118); the vitamin B, -dependent oxidative decarboxylation of pyru-
92 Vitamin B,
Vitamin B^ and Carbohydrate Metabolism
Dietary Glycogen Pentose phosphate

Carbohydrates Cycle
Glucose-6-phosphate
Lipids
(Triglycerides) Fructose-6 -phosphate
Ribulose- 5 -phosphate
a-Glycero-
phosphate 3 -Phosphoglyceraldehvde Ribose-5 -phosphate
N
Lactate Pyruvate Nucleotides
Vit.B,
n1/
Acetyl-CoA
(Active Acetate)
Oxalacetate
a-Ketoglutarate
CO.
\'itamin Bj 93
vate thus leads, through this intermediate, to the complete degradation of

carbohydrates. Active acetate is also the fundamental building unit for fatty
acids and steroids, so that vitamin Bj is indispensable for the transformation
of carbohydrates to lipids.
The accumulation of pyruvate and lactate appearing in blood and tissues
in vitamin Bj deficiency is due to reduced activity of the pyruvate dehydro-
genase complex. In this connection mention may be made that through its
vitamin B, -dependent conversion to acetyl-coenzyme A, pyruvate also

provides the acetyl group for acetylcholine involved in nervous trans-
mission. Thus, a deficiency of vitamin Bj affects acetylcholine levels in the
brain. Whether, and to what extent, vitamin Bj participates directly in the
excitation of peripheral nerves other than as a coenzyme requires further
clarification.
The oxidative decarboxylation of a-ketoglutarate, catalysed by vit-

amin Bj with formation of succinyl-coenzyme A, is an essential component
reaction of the citric acid cycle, the normal functioning of which enables
an optimal utilisation of energy liberated in the oxidative final degradation
of acetate.
Further, vitamin Bj, as the coenzyme of transketolase, takes part in the
so-called pentose phosphate cycle which facilitates the cell's conversion
of pentose phosphate to hexose phosphate. Vitamin B, thus fulfils an
important function bv channelling the pentoses into the energy-yielding
oxidative degradation of the hexoses. The further significance of the pentose
phosphate cvcle in general cellular metabolism Hes in making the pentoses
available for the synthesis of nucleotides and nucleic acids and in the forma-
tion of reduced nicotinamide adenine dinucleotide phosphate (XADPH,),
required for fatty acid synthesis in the cell.
As vitamin B, is so intimately bound up with carbohydrate metabolism,

a diet rich in carbohydrate increases the normal requirement for vitamin Bj.
General Deficiency Sjwpfoms
Severe deficiency of vitamin Bj can show itself in ff/an or in different

animal species^ according to living conditions, in two major groups of
symptoms, brought about by cardio-vascular damage and nervous dis-
orders, respectively. The first group includes oedema due to damaged
arterioles and pre-capiUaries and heart disorders which appear, for example,
as shortness of breath, tightness of chest, and tachycardias and can cause
sudden death. The second group includes hyper- and hyposensitivity,
burning feet, neuritis, muscular weakness and pain and spasms extending
to paralyses.
In a severe vitamin B, deficiency one or both of these groups appear in
pronounced form (beri-beri in man); this is not so in early stages of de-
; ;
94 Vitamin B,
ficiency, particularly if other symptoms are superimposed. Neuraesthenia,

fatigue and disturbed emotional equilibrium should be regarded as signs
of latent vitamin B, deficiency; to these may be added diminished appetite
and disordered digestion (diarrhoea, intestinal atony, achlorhydria).
In ruminants with a functionally developed rumen in general the vit-
amin Bi requirement is covered by bacterial synthesis in the rumen.
However, sporadic outbreaks of Bj-deficiency do occur in adult ruminants
when kept on rations high in digestible energy.
General condition Decrease and loss of appetite, inhibition of growth,

weight loss, general weakness, lowered tempera-
ture, progressive weakness, dyspnoea (pig, rat,
mouse, dog, chicken, turkey-hen).
Skin and mucous Cyanosis (chicken, pig);

membranes of the head Lacrymation (calf).
Nervous system Polyneuritis, ataxia, spastic pareses and paralyses

(e.g. Chastek's paralysis of foxes), later, spasm
and convulsions (fox, mink, calf, sheep);

Opisthotonus (head retraction) (pigeon, chicken,
fur bearing animals, calf, lamb);
"Stargazer-disease" (lion).
Blood and vascular Slowing of pulse (pig, rat) and respiration (rat);
svstem Bradycardia, heart dilatation and fatty degenera-

tion of heart muscle fibres (pig)
Cyanosis, oedema (pig, chicken).
Gastro-intestinal Diarrhoea and secondary dehydration, intestinal

tract atony, achlorhydria, (rat, mouse, pig);
Gastro-intestinal haemorrhages (pig).
Reproductive system Inhibition of testicular development (cockerel)

Ovarian atrophy (hen);
Premature births and high mortality of new-born
(pig)-
Other symptoms and Transketolase test;

diagnostic tests Raised reactivation values of transketolase after
addition of cocarboxylase to erythrocyte haemo-
lysate in vitro (rat, man etc.).
Vitamin B, 95
General weakness,
cyanosis and poly-
neuritically induced
symptoms of para-
lysis mark the
outward appearance
of vitamin B,
deficiency in a
chick.
Human Requirements
The requirement for vitamin B, follows from its function of regulating

the carbohydrate metabolism; it increases with the carbohydrate content
of the diet. The minimal requirement of adults is 0.5 mg vitamin B, per
1000 calories; but with a dietary intake of less than 2000 calories per day,
the vitamin supply should not fall below i mg per day. Conditions which
increase metabolism, such as heavy labour, pregnancy or feverish illness
demand an increased supply of vitamin Bj. A high urine excretion increases
the vitamin excretion and, therefore, the vitamin requirement. Gastro-
intestinal disturbances are frequently accompanied by reduced resorption
by the intestinal wall so that the vitamin supplied is only partly taken up.
The great importance of the vitamin during the growing period is
indicated by the relatively high requirement, for example, of six month

old infants namely 0.27 nig/iooo cals. or 0.14-0.20 mg per day. Because of
the relatively low vitamin Bj content of the mother's milk (0.15 mg/I=
0.21mg/iooo cals.) a daily dose of 1.5 mg is recommended for the lactating
mother. However, according to other data, the optimal supply of vitamin Bj
for suckling infants is only assured if the mother's intake is 4. 5 mg vitamin
B,.
Vitamin B:
The component features in the structure of vitamin B2 are the flavin

pigment lumichrome and the reduced form of the sugar ribose, giving rise
to the name riboflavin; the synonym lactoflavin relates to its occurrence
in milk, where its presence is shown by a yellow-green fluorescence in the
whey.
Riboflavin is present in virtually all living cells. It occurs most usually
in the form of a dinucleotide or a phosphate ester, or it may be bound to
a protein. In the free state, significant amounts of vitamin B2 are reported
to occur only in milk, urine or retinal tissues. Foods that are important
sources of vitamin B, are liver, kidney, meat, fish, yeast, milk, cheese, eggs
and vegetables.
Because of the low solubility of riboflavin in water, the water-soluble
sodium riboflavin-5 '-phosphate has become an important form of the vit-
amin.
Structural formulae CH2 — (CH0H)3— CH2R
.ONa
Riboflavin: R=OH; Sodium Riboflavin-5 '-Phosphate R = -0-P^
\OH
O
Empirical formula Riboflavin: Ci7H2oOf,N4
5
Sodium Riboflavin- '-Phosphate: Ci7H2oOgN4PNa
Molecular weight Riboflavin: 376.4

5
Sodium Riboflavin- '-Phosphate: 478.4
Description Yellow to orange-yellow crystalline powder.
SolubiUty Riboflavin: Readily soluble in dilute alkalis (de-

comp.) sparingly soluble in water (ca. 7 mg/
1 00 ml), very sparingly in alcohol, insoluble in ether
and chloroform.
Sodium Riboflavin-5 '-Phosphate Soluble in water, :
very sparingly soluble in alcohol, insoluble in

ether, acetone and chloroform.
Vitamin B, 97
Melting point Decomposition at ca. 280-290 °C
Specific rotation Riboflavin:

[a]^° = -i22° to -156° (c=o.25 in 0.05 N.NaOH.
5
Sodium Riboflavin- '-Phosphate:
[a]^°= +38° to +42° (c = i.5 in 20% HCl).
Absorption spectrum o.iN HCl solutions of riboflavin and the phos-

phate show absorption maxima at ca. 223, 267,
374 and 444 nm.
Stabilitv Both compounds are sensitive to light and U.V.

radiation, but are stable to heat and atmospheric
oxygen. In alkaline solutions decomposition is
fairly rapid, especially if exposed to light.
The
assay of vitamin B, by direct fluorometry of the intact compound
is most widely used procedure. Also of widespread use is the lumiflavin
the
method which takes advantage of the fact that chloroform insoluble ribo-
flavin yields chloroform soluble lumiflavin upon irradiation in alkaline
solution. As good separation and enrichment procedures for the vitamin
are available, such as, e.g., chromatography on talcum columns, permutits
or Fuller's earth, it is sometimes possible to use direct photometric measure-
ment of the yellow colour of riboflavin.
Colorimetric reactions, such as those with mercuric sulfate, silver nitrate
or cupric chloride triphenyl phosphine complex, are seldom used.
Several polarographic techniques are available which are very simple
and reliable but which sometimes also require the removal of interfering
substances.
For low potency samples, foods and feeds, the microbiological test is,
however, still in use. The microorganism used is lactohacillns casei.
No International Unit of vitamin B, has been defined. The biological

activity of preparations containing vitamin B-, can be examined in the curative
rat growth test, crystalline riboflavin being used as reference substance.
An out-of-date definition of biological activity is: i mg riboflavin is
equivalent to 250 rat Units or 50 Sherman-Boiirquin Units.

c)8 Vitamin B2
Vitamin B,, in the form of the phosphate ester (flavin mononucle-

otide=FMN) or of the flavin adenine dinucleotide (=FAD), acts as a
component (coenzyme) of the flavin enzymes concerned with hydrogen
transfer. Above all, the role in the respiratory chain should be noted.
The enzymes NADH^- and NADPH^-cytochrome c-reductase
flavin
and also succinic dehydrogenase transfer hydrogen from reduced nicotin-
amide" adenine dinucleotide (NADH,) and nicotinamide adenine dinu-
cleotide phosphate (NADPH2) or from succinate, with concomitant reduc-
tion of the coenzyme. In the subsequent oxidation of the flavin enzymes
by cytochrome, the hydrogen electron is taken up by the iron of cytochrome
and the hydrogen ions then form water with oxygen ions.
In the wider sense the flavin enzymes of the respiratory chain intervene
in the general metabolism of carbohydrates, amino acids and fatty acids in ;
the oxidation of the numerous substrates thev take up hydrogen from the
various specific substrate dehydrogenases and provide for the oxidation
to water (see also Nicotinic acid, p. 1 1 8).
Further flavin enzymes with roles in intermediary metabolism are the

D- and L-amino acid oxidases which dehydrogenate the amino to the corre-
sponding imino acids. The latter break down spontaneously to a-ketoacids
and ammonia. The reduced D- and L-amino acid oxidases and xanthine
oxidase transfer the hydrogen directly to molecular oxygen and are them-
selves reoxidised in the process.
Xanthine oxidase effects the oxidation of hypoxanthine and xanthine
to uric acid and of aldehydes to the corresponding carboxylic acid. In
addition, vitamin B2, as a component (FAD) of acyl-coenzyme A dehydroge-
nases, plays a part in the degradation of saturated fatty acids, the dehydro-
genation of which is catalysed by these enzymes. This dehydrogenation is
Vitamin B-^ and the Respiratory Chain
Substrate-(lH2) Succinate- (HJ @ = Elearons

' ^
i Flavin enzymes
NAD-(H^ Vit.B. —(h^ —^Cytochrome @
system ^Cytochrome (e)
ox idase Ôxygen
;
Vitamin B2 99
an important intermediate stage in the degradation of the activated fatty

acids bv ^-oxidation with the elimination of acetate. In the opposite sense
vitamin B2 is also required, in the form of FMN, as the coenzyme of de-
hvdroacvl reductase in the synthesis of long-chain fatty acids from acetate.
This role of vitamin B2 in fatty acid metabolism provides an explanation of
the increase of normal vitamin B, requirement on a high-fat diet.
While vitamin B^ is present in most animal organs as FMN or FAD,
the lens, retina and cornea of the eye contain free vitamin B, in relatively
large amounts. So far, however, nothing precise is known of the actual
function of vitamin B, in the eye, or of a possible role in the visual process.
In vitamin B, deficiency vascularisation of the cornea and clouding of the
refractive media are observed.
General Deficiencj Symptoms
Severe riboflavin deficiency is generally not recognisable as a marked

and characteristic pathology but shows itself in several non-specific symp-
toms. The most important of these is fatigue and work, changes
inability to
in the lips, the buccal mucosa and tongue, in the horny skin and also in the
rest of the bodv skin (in man especially of the eyes, nose, tongue, gastro-
intestinal tract, anus, vulva and scrotum).
The fall in feed utilisation in vitamin B, deficiency is a fact of special
importance in animal husbandry; endogenous protein synthesis is hindered
by inadequate amino acid oxidase action, so that part of the dietary intake
of amino acids is excreted in the urine.
Deficiency of vitamin B2 during pregnancy leads to skeletal foetal ab-
normalities (shortened bones, deformed growths between ribs, toes and
fingers, brachydactyly).
A further symptom of vitamin B, deficiency is reduced sharpness of
vision and rapid eye fatigue.
In less severe deficiency of vitamin B, all the described symptoms may
appear, albeit in less marked form; further, a latent or overt vitamin B,

deficiency is usually accompanied by symptoms of other nutritional in-
adequacy, so that recognition is diflficult.
Deficiencj Symptoms in Animals
General condition Slow or halted growth, reduced appetite (pig, dog,

mouse, chicken, turkey-hen)
fox, rat, guinea-pig,
Fall in body temperature (dog).
; ;
\'itamin B,
Skin and mucous Seborrhoeic inflammation of buccal and nasal

membranes of the head mucosae, corner of the mouth and eye-lids, com-
bined with copious tears and salivary flow;
Dry, scaly dermatitis, loss of hair, rough coat (pig,
cat, rat, calf, lamb);
Bleaching of fur colouring (fox)
Skin inflammation of extremities, encrusted beak,
(turkey-cock).
Eve Light sensitivity, lens turbidity (pig, dog, fox,

cat, trout);
Vascularisation of cornea, cataract (rat);

Conjunctivitis (horse).
Nervous system Spasms and flaccid paralyses, ataxias due to myelin

degeneration of peripheral nerve bundles, pyrami-
dal tracts and cranial nerves (birds, mammals)
Curled toe paralysis, sitting with outstretched legs
(chicken).
Digestive tract Hiccough, vomiting, resorption disorders and

diarrhoea due to inflammation of mucous mem-
branes of digestive tract (pig, chicken, poultry).
Vitamin B2 defi-
ciency in the pig:
dry, scaly dermatitis
with loss of bristles,
inhibited growth
and loss of appetite.
;
\'itamin B,
2-Week old chick

with symptoms of
vitamin Bj deti-
ciency: curled toe,
paralysis of ex-
tremities.
Reproductive svstem Disturbance of egg-laving, reduced hatchability of

eggs (high embrvo mortalitv in second incubation
week), stunting and extensive oedema, "clubbed"
down of embryos (poultry). Resorption sterility,
premature and still births (pig)

Oedema (piglet).
Other svmptoms and Glutathione reductase test:
diagnostic tests Increased activity of NADPH,-dependent gluta-

thione reductase after addition of flavin adenine
dinucleotide (FAD) to whole blood or ervthrocvte
haemolysate in vitro (rat, man, pig, chicken etc.)
Human Requirements
In accord with the function of vitamin B,, the requirement for this
vitamin is related to the metabolic rate ; it also appears to be related to body
weight. The dailv human requirement varies between i and 3 mg, depend-
ing on age and living conditions. Normally 1.1-1.6 mg per day suffice for
an adult; below 0.6 mg per dav deficiency states appear. The minimum
required bv a child is 0.4-0.5 mg per day. The requirement increases
in pregnancv, during lactation and growth, and in sickness such as infec-
tions, high thvroid activitv etc., as well as for increased intake of fluids.
Vitamin B6
Vitamin Bg activity is shown by three substituted pyridine derivatives

which only differ in the functional group in the 4-position; these are the
alcohol pyridoxine or pyridoxol, the aldehyde pyridoxal and the amine
pyridoxamine. All three compounds are often considered together as pyri-
doxine. As pyridine derivatives, they all form salts.
Since the analytical detection of substances with vitamin B^, activity is
very difficult, only incomplete data are available on its natural occurrence.
Sources of vitamin B^ in human nutrition are red meat, liver, kidney, brain,
cod-liver and roe, egg-yolk, milk, yeast, cereals and green vegetables.
The commercial preparation of vitamin B^ is almost exclusively of the
hvdrochloride of the alcohol (pyridoxine hydrochloride).
Physico-chemical Properties (pyridoxine hydrochloride)
Structural formula
CH2OH
HO^ ..^^^ ^CH20H

•HCI
CH3 N
Empirical formula CgHjiOjN.HCl
Description White crystalline powder
Solubility Readilv soluble in water (ca. i g/5 ml), sparingly

soluble in alcohol, insoluble in ether and chloro-
form.
Melting point Decomposition with browning at ca. 206 °C.
Absorption spectrum In aqueous solutions, the absorption maxima are, for

acid pH, at 291 nm, for neutral at 254 and 524 nm
and for alkaline solutions at 245 and 309 nm.
Stability Pyridoxine hydrochloride is stable to heat and

oxygen. Pyridoxine is degraded by light in alkaline
or neutral solution and, to a lesser extent, in acid
solution.
Vitamin Be 103
As already stated, vitamin B^ may occur in three different forms (pyri-
doxine, pvridoxal and pvridoxamine) and if it is not clear that one of these
is present almost exclusively in a preparation containing vitamin B^, there
is difficulty in its assay. Pharmaceuticals and enriched dietary products
nearly always contain pyridoxine, while the possible presence of all three
forms may be expected in foods and feedstuffs that have not been enriched.
In pure solution and high potency products of simple composition,
pyridoxine mav be assayed by titration with perchloric acid in non-aqueous
medium, bv titration with sodium thiosulphate after oxidation with mer-
curic aceate or bv U.V. photometric measurement of the difference in absor-
bance of the compound at different pH.
The coloured reactions obtainable with the vitamin B^, substances have
with those of para-unsubstituted phenols. A reddish brown
similarities
colour forms with ferric chloride, a blue colour is obtained with phos-
photungstic or phosphomolybdic reagent. Orange or red colours are formed
with diazotized p-aminoacetophenone or nor-sulfazol and zinc
sulfanilic acid,
chloride. Blue indophenol derivatives are formed with dichloroquinone

chlorimide and a similar reaction occurs with N,N-diethyl-p-phenylendi-
amine in the presence of an oxidant. A cyanine dye is obtained by treatment
of pyridoxine with diazomethane, N-methylation and condensation in the
presence of chloroform and alkali.
Colour reactions only given by pyridoxal are the condensation with

acetone in the presence of a base to a yellow product, the reaction with
thiophene to a green derivative and a yellow colour produced with sulfuric
acid.
Pvridoxamine is ninhvdrin positive; it may also be transformed to
pyridoxal by transamination with glyoxylic acid.
These colour reactions may be considered for the determination in
highly concentrated products of known composition, but they lack sensi-
tivity for biological material.
Fluorometric determinations have a higher sensitivity but they do not
solve the problem of determination in biological samples. The three com-
pounds fluoresce in neutral solutions, however the natural fluorescence
is weak. Better sensitivity is achieved by oxidizing pyridoxine or pyridoxal
with potassium permanganate to 4-pyridoxic acid, whose lactone exhibits
strong fluorescence in alkaline medium. Pvridoxamine may be converted
to pyridoxine with nitrous acid and subjected to the same reaction sequence.
Unfortunately, the recoveries are low and differ between the three forms.
Alternatively, pyridoxal may be reacted with potassium cyanide to a
highly fluorescent derivative.
Combinations of chromatographic separation methods with the fluoro-
metric determinations mav be used sucessfullv for enriched foods and feeds.
I04 Vitamin B^
Equally good results are obtained using gas-liquid chromatography.

Pyridoxal may also be determined by polarography.
The evaluation of the vitamin B^ activity of non enriched material such
as foods and tissues is still made predominantly by microbiological methods.
Suitable test organisms are neiirospora sitophila or saccharomyces carlshergensis.
With the microbiological methods, it is possible to differentiate between
pyridoxine, pyridoxal and pyridoxamine by suitable selection of micro-
organisms. Thus, neiirospora sitophila and saccharomyces carlshergensis respond
more or less equally to all three vitamin B^ forms, while another micro-
organism, streptococcus faecalis responds to pyridoxal and pyridoxamine and
a further microorganism, lactohacillns casei, responds almost exclusively to
pyridoxal. Assay of a sample with all the above organisms provides in-
formation about the relative amounts of the three components.
No International Units of vitamin B(, have been defined. Analytical

results are expressed in weight units of pyridoxine hydrochloride.
I mg Pyridoxine hydrochloride is equivalent to

0.82 mg Pyridoxine (Pyridoxol)
0.81 mg Pyridoxal
0.82 mg Pyridoxamine
The biological activity of preparations containing vitamin B^, can be

assayed with a growth test in the rat or chick. An earlier method is based
on assessment of healing of skin lesions in vitamin B^-deficient rats (rat
acrodynia test). The smallest daily -dose capable of healing rat acrodynia
was termed a "Rat Unit" in the past; it corresponds with the activity of
ca. 7.5 meg pyridoxine hydrochloride.
In the form of pyridoxal 5 '-phosphate, vitamin B5 acts as the coenzyme

of a series of enzymes which catalyse transamination, decarboxylation,
deamination and desulphydration, and the cleavage or synthesis of amino-
acids. The aminotransferases transfer amino groups between amino acids
and a-ketoacids for example from glutamate to pyruvate with formation of
;
a-ketoglutarate and alanine, or from aspartate to a-ketoglutarate, forming

oxalacetate and glutamate. The aminotransferases thus represent an im-
portant Link between amino acid, carbohydrate and fatty acid metaboUsm
and the energy-producing citric acid cycle.
Vitamin B(, 105
Vitamin B^ and Amino acid Metabolism
Metabolism of Proteins
and Amino acids
r^
Vit. B6 Vit. B6 Vit. Bfi Vit. B6
\/ Vit. Eft
Alanine Pyruvate Carbohydrate
"^ Active Acetate -^ Fatty acid

Lipids
Citrate
Vit. Bo
Aspartate are Oxalacetate
Vit. B6
Glutamate !a-ketoglutarate
lo6 Vitamin 65
The decarboxylases convert amino acids to the corresponding biogenic

amines, such as histamine, hydroxytyramine, serotonin, y-aminobutyric acid,
ethanolamine and taurine, some of which are substances of high physio-
logical activity (regulation of blood vessel diameter, neurohormonal actions,
essential components of phospholipids and bile acids). In accord with their
general importance these enzymatic reactions take place in virtually all
organs, most intensively in the liver, heart and brain. Other enzymatic
reactions (deamination, desulphydration) which are more related to the
catabolism and anabolism of amino acids are localised mainly in the liver.
Abnormal tryptophan metabolism due to vitamin Bf, deficiency can be

used for diagnosis because it appears at a very early stage of deficiency,
giving rise to increased urinary excretion of xanthurenic acid, especially
after a test dose of tryptophan. The increased formation of xanthurenic
acid occurs because kynureninase activity is more markedly restricted by
vitamin B^, deficiency than is kynurenine a-ketoglutarate aminotranferase.
Finally, normal amino acid metabolism is also highly important for
detoxification reactions which eliminate harmful substances from the body.
Adequate provison of vitamin B^ is also required for maintenance of a
normal level of coenzyme A in the liver. The biosynthesis of coenzyme A,
essential for fatty acid metabolism, is impaired in vitamin B^ deficiency,
from which disordered lipid metabolism can result. Conversely, an increased
dietary intake of fat can result in lowering of the pyridoxal phosphate level
in the liver. Probably the increased ingestion of fat raises the body's B2
requirement in ; this way the activity of the vitamin B2-dependent pyridoxal
phosphate oxidase, responsible for the formation of pyridoxal 5 '-phosphate,
is reduced.
The intimate involvement of vitamin B^, in amino acid metabolism
readily explains why an increase of dietary protein leads to increased vit-
amin B5 requirement.
Severe vitamin B^ deficiency showsitself in man or in different animal
depending on age and other conditions, by the appearance of skin

species,
changes (in man, seborrhoeic dermatitis in the areas of the nose, eyes and
mouth, erosions of the buccal mucosa and mouth, glossitis) or nervous
disorders (peripheral neuritis, especially sensitivity disorders in man). The
latter are primarily seen during the period of active growth (epileptiform
convulsions in the infant).
In mild vitamin B^ deficiency a number of non-specific symptoms
appear, which resemble those that can result from deficiencies of other
vitamins of the B group. The disordered protein metabolism and associated
;; ; ; ; ;
Vitamin Br, 107
deficient feed utilisation in vitamin B^ deficiency are of particular importance

in livestock management.
Deficiency Symptoms in A.nimals
General condition Diminished appetite, slow growth, poor feed

(pig, dog, rat, guinea-pig, mouse,
utilisation
poultry, turkey, duck).
Skin and Flaking and thickening of the skin of the ears,

mucous membranes severe inflammation and encrustation of eyes,
of the head nose, paws and tail (=acrodynia) (rat, rabbit);
Rough coat, brown conjunctival exudate (pig);

Loss of hair (calf, rabbit)
Greying of fur, oedema of eye-lids, fissures of the
palms of hand and foot (monkey)
Rough, deficient plumage (poultry);
Blue-green discolouration of the back (trout).
Eyes Disordered vision (pig, trout).

Nervous system Disordered hearing, ataxia, increased irritability,
epileptiform convulsions, paresis, general weakness,
demyelinisation of peripheral nerves (rat, mouse,
dog, pig, calf, cat, rabbit, poultry, turkey, trout).
Blood Microcytic hypochromic anaemia (pig, dog, poul-

try, turkey, duck, rat, trout, salmon);
Increased iron content of plasma (dog, pig).
Digestive tract Fatty infiltration of liver (pig)

Loss of appetite, diarrhoea (pig, poultry, trout,
calf);
Stomach ulcers (poultry).
Reproductive Fall in egg production, lowered hatchability, (high

system embryonic mortality in second week) (poultry,
turkey)
Reduced fertility (mink bitch), sterility (male mink).
Other symptoms Ascites (accumulation of serous fluid in abdominal

and diagnostic tests cavity)
Rapid, gasping respiration (trout);
Ervthrocvte-GOT*-test
*GOT=Glutamate oxalacetate aminotransferase

io8 Vitamin Be
Inflamed oedema
of the eyelids in
vitamin B^ defi-
ciency (Poultry).
Vitamin B(, defi-
ciency in poultry;
rough, deficient
plumage, weakness
and inco-ordination
of movements.
;
Vitamin B^ 109
Piglet with severe

disruption of
growth and der-
matitis resulting
from vitamin 65
deficiency.
Increased activity after pyridoxal 5 '-phosphate

addition to the erythrocyte haemohsate
Increased xanthurenic acid excretion after trypto-
phan loading dose.
Human Requirements
In its phosphorylated form vitamin B,, has the role of coenzyme for
numerous enzymes and is essential, for example, in the metabolism of
protein. The vitamin B^ requirement therefore increases with the level of
protein intake and is estimated as between i and 2 mg per day for humans.
This is increased during pregnancy (ca. 10 mg per day).
Vitamin Bi
The compounds active as vitamin Bj, are of complicated chemical

structure; the best known and most important is cyanocobalamin, the others
only differing slightly from this.
Vitamin B12 occurs only in animal material and in metabolites of micro-

organisms. The synthesis of this vitamin in the alimentary tract is of con-
siderable importance for animals ; if sufficient cobalt is available, ruminants
are independent of external sources of vitamin Bj^. Important sources of
vitamin B12 in human nutrition are liver, kidney and egg-yolk.
Physico-chef?iical Properties
Structural formula
NH2 — CO — — CH2 CH2 CH3 CH3 CH2 — CO — NH2

NH2— CO — CH2 — CH2 — CO —
CH2 NH2
CH3
CHs
NH2 — CO — CH2 CH3
CO — CH2 — CH2 CH3 CH3 CH2 — CH2 CO NH2
NH
I
CH2
OH — CH2
Empirical formula CejHggOi^Ni^PCo
Molecular weight 1 3 5 5 -4
Description Dark red crystalline powder

—
Vitamin B,2
Solubility Slightly soluble in water g/8o ml), soluble

— ^^ alcohol, insoluble in ether
(ca. i
and chloroform.
Melting point Cyanocobalamin chars at 210-220 °C without pre-

vious melting.
Absorption spectrum Aqueous solution shows absorption maxima at
ca. 278, 361 and 550 nm.
Stability Crystalline cyanocobalamin and its neutral to

weakly acid solutions are relatively stable to air
and heat, but are attacked by light and UV radia-
tion. Vitamin B,, is only slightly stable to alkalis.
strong acids and reducing agents.
Methods of Determination in Simple Media
High potency preparations of vitamin Bj, are usually assayed by spec-

trophotometry in the U.V. or in the visible part of the spectrum. Some
procedures use highly efficient purification techniques by ion exchange,
partition or adsorption chromatography, so that a direct measurement at
one or two wave-lengths is satisfactory. Other procedures eliminate the
bulk of interfering substances by benzyl alcohol extraction of the dicyanide
complex of vitamin B12 and measure the difference in absorbance between
the mono and dicyano form which is independent of background absorb-
ance.
As one mole of vitamin B,^ will liberate one mole of hydrocyanic acid
upon irradiation with U.V. light or reductive splitting, the very sensitive
assay of hydrocyanic acid, with chloramine-T and pyrazolene pyridine
reagent, yielding a blue dye may also be used for the determination of the
vitamin.
Alternatively, vitamin B12 assays may be based on the determination
of cobalt, which is quantified by colorimetry with nitrose-R salt, atomic
absorption spectroscopy or by radiometric methods. In the latter, the cobalt
arising from vitamin B,, is labelled with a trace of 60 Co and bound to a
cation exchanger. The on the resin is counted before and after
radioactivity
equilibration with a sub-stoichiometric amount of ethylene diamine tetra-
acetic acid, which removes an equivalent amount of cobalt from the ex-
changer. The difference between the two radioactivity measurements thus
becomes a measure of the dilution of the tracer Co and hence of the total
cobalt present.
A further technique is the polarographic determination, which is, how-
ever, not common.
Vitamin B,2
Since vitamin B,, occurs at extremely low levels in natural products,

concentration and purification steps are essential. The vitamin is always
accompanied by its decomposition products or by compounds related to
its structure which usually interfere with the determination. For the purifi-
cation of vitamin B,, extracts, a variety of column, paper or thin-layer

chromatographic methods and combinations with electrophoretic and ion
exchange procedures are available. Losses occurring during purification are
assessed using radioactive vitamin B,, as internal standard.
As the final evaluation by the methods mentioned above is seldom
possible, the microbiological assay is still used.
The four best known test organisms for vitamin B,^ are the two flagellates
euglena gracilis var. hacillaris and ochromonas malhamensis, and among the
bacteria, lactohacillus leichmannii and certain mutants of escherichia coli. As the
sensitivity of the different organisms to the active individual compounds
of the Bi2 complex is not the same, parallel determinations with two different
microorganisms are sometimes necessary.
The present trend is to replace the microbiological assay by methods
using the isotope dilution principle. These radiosorbent methods use a
specific binder protein in sub-stoichiometric amounts, to which the vit-
amin Bj, of the sample and the added radioactive vitamin Bj, bind. The
total binding capacity and the added radioactivity being constant, the
amount of radioactivity remaining unbound becomes a function of the
dilution of the label by the vitamin from the sample and hence a measure
of the unlabelled vitamin Bj^ present.
No International Units have been defined for the biological activity

of vitamin Bj^. Pure cyanocobalamin can be used as standard substance.
Such a preparation is available from the WHO.
For the biological test of preparations containing vitamin Bj, the clinical
experiment is used. The daily dose giving clinically and haematologically
satisfactory results in authentic pernicious anaemia provides the criterion.
Less commonly used definitions of the biological activity are: i meg
cyanocobalamin is approximately equivalent to ii,ooo LLD-Units (lacto-
hacillus lactis Dorner) or i U.S. P. liver-extract Unit.

^'itamin B,2 1 1
3
Vitamin Bj, exerts an effect reactions. Above all

on various metabolic
it is necessary in the reduction of one-carbon compounds of the oxidation
stage of formate and formaldehyde, and in this way it participates, with
folic acid, in the biosynthesis of labile methyl group s. The formation of the
latter is required for the biosynthesis of purine and pyrimidine bases, the
essential components of the nucleic acids (see Folic acid, p. 141). Apart from
this, the metabolism of labile methyl groups plays an important role for
the body in the biosynthesis of methionine from homocysteine and of
choline from ethanolamine. Methionine functions on the one hand as an
indispensable building unit for proteins and on the other as a methyl group
donor for the biosynthesis of Upotropically active choline and for the forma-
tion of creatine which serves, after conversion to creatine phosphate, for
energy storage in muscle tissue.
Folic acid is also essentially involved in all these reactions of labile
methyl groups. According to present knowledge there aremainly two points
of connection between vitamin Bj, and the one-carbon compound metab-
olism dependent on folic acid. On the one hand a loss of activity of hydroxy-
methyl-tetrahydrofolic acid dehydrogenase is observed in vitamin Bjj
deficiency, so that it may be assumed that vitamin Bj2 is a cofactor of this
enzyme system. On the other, there are grounds for the view that vitamin B12
is necessary for storage of folic acid in the liver.
A further important function of vitamin B,, in intermediary metabolism
consists in maintaining glutathione and sulphydryl groups of enzymes in
the reduced state. The fall in activity of glvceraldehyde 3-phosphate dehydro-
genase, which requires glutathione as coenzyme, is possibly responsible for
the impairment of carbohydrate metabohsm in vitamin B,2 deficiency. The
influence of vitamin B,, on lipid metabolism probably follows, similarly,
from its on thiols. A deficiency of this vitamin also induces a loss in
action
activity of methylmalonyl-coenzvme A isomerase, which participates in the
transformation of propionate to succinate. As a consequence there is a
sharp increase in the urinary excretion of methylmalonic acid; this can be
used for the biochemical diagnosis of vitamin Bj, deficiency
at an early stage.
Absorption of vitamin B,, in the digestive tract reqtiires "I ntrinsic
Factor" a mucoprotein secreted bv mucosal cells of the stomach (man) or
,
by the pyloric and duodenal mucose glands (pig).
In man vitamin B,, deficiency usually only occurs if insufficient vitamin

isabsorbed due to pathological conditions (inability to produce "Intrinsic
Factor", gastrectomy, tape-worm infestation).
;
114 Vitamin B12
Vitamin B12 defi-

ciency in the pig:
smaller size and
dishevelled coat in
contrast to normal
animal (lower
picture).
In animals a nutritionally induced deficiency of Bj, is possible, especially

if an exclusively vegetable feed is given or if the vitamin synthesis by in-
testinal flora is not assured, due to lack of cobalt in the feed of ruminants
such deficiency shows itself primarily by poorer feed utilisation and asso-
ciated impairment of protein deposition.
Advanced deficiency leads to a change in the blood picture (human
pernicious anaemia) and to certain nervous disorders (axon degeneration.
of the nerves of the spinal cord) . In man these conditions can only be
ameliorated with difficulty by vitamin Bj, administration.
General condition Reduced growth, poor feed utilisation (pig, dog,

rat, mouse, chicken, turkey).
Skin Rough coat, dermatitis.

;
Vitamin Bi^ 115
Nervous system Increased irritability, loss of yoice, unco-ordinated

moyements, painful hind-quarters (pig);
Ataxia, demyeUnisation of peripheral nerves (calf,
on vitamin B,,-free milk-replacer diets).
Blood Mild to medium grade normocytic anaemia (pig,

mink, fox, trout).
calf,
Digestive tract Loss of appetite, diarrhoea and vomiting (pig).
Reproductive system Reduced hatchability of eggs (embryonic deaths

week) (chicken, turkey)
in the last
Diminution in size and weight of litters (pig);
Reduced viability and high mortality of young
(fur-bearing animals).
Human Requirements
The exact daily human requirement for vitamin Bj, cannot be given,
particularly as it is s ynthesised by intestinal flora. It appears to be about
2 meg. In the absence of "Intrinsic Factor" the vitamin is not absorbed.
Nicotinic Acid and Nicotinamide
Nicotinic acid (niacin) and nicotinamide (niacinamide) possess the same

vitamin activity; the free acid is converted to the amide in the body. The
less commonly used synonyms vitamin PP and PP-factor refer to their
pellagra preventive activity.
Nicotinamide occurs in all living cells, usually in a bound form as the
prosthetic group of coenzymes. Liver and meat of hoofed animals are

nutritionally important sources of this vitamin. It is also present in maize
and other cereals in a from not utilised by man.
"OH NH?
Structural formulae
Nicotinic Acid Nicotinamide
Empirical formula Nicotinic Acid: C^HjO^N

Nicotinamide: QH6ON2
Molecular weight Nicotinic Acid: 125.1

Nicotinamide: 122.1
Description White crystalline powders
Solubility Nicotinic Acid: Sparingly soluble in water (ca.
I g/60 ml) and alcohol (ca. i g/ioo ml), readily

soluble in alkalies, insoluble in acetone and ether.
Nicotinamide: Very soluble in water (ca. i g/ml)
slightly in alcohol, soluble in glycerol, sparingly
soluble in ether and chloroform.
Melting point Nicotinic Acid: 234-237 °C (Sublimation)

Nicotinamide: i28-i3i°C
Absorption spectrum The acid and amide show similar absorption spec-
tra inaqueous solution with a maximum at ca.
261 nm, with an extinction dependent on pH.

Nicotinic Acid and Nicotinamide 117
Stability Both compounds are stable to atmospheric oxvgen,

light and heat in the dry state and in aqueous solu-
tion; the amide is hvdrolysed to the acid bv heating
in strong-lv acid or alkaline solution.
Methods of Deter f?iination in Simple Mixtures
The majority of the photometric methods for the assav of nicotinic

acid and nicotinamide utilise the Konig-reaction. Nicotinic acid or nico-
tinamide is cyanogen bromide which breaks one carbon
treated with
nitrogen linkage of the pyridine ring.The glutaconedialdehvde formed is
reacted with an aromatic amine to vield a polvmethin dve which can be
determined colorimetrically. Amines which have been used are barbituric
acid, sulphanilic acid, p-aminoacetophenone, m-phenylene diamine and
others. The reaction with barbituric acid has a good selectivitv for nico-
tinamide. Nicotinic acid, a possible degradation product in preparations
containing nicotinamide, does not respond to the reagent. Other techniques
are U. V. photometry, colorimetry of the complex with bromothvmol blue
or of the derivative with sodium i,2-naphthoquinone-4-suifonate. Nicotin-
amide may also be transformed to 5-aminopyridine by sodium hvpobromite,
diazotised and coupled with N-phenvl-/3-naphthvl-amine.
Most photometric assays include chromatographic purifications to
avoid interferences. Several thin-laver chromatographic and ion exchange
techniques for polyvitamin preparations have been described.
Non photometric techniques are the direct titration with perchloric acid
and the polarographic assav.
in glacial acetic acid
The determination by gas-liquid chromatography is also possible.
Fortified foods or feeds are usually assayed by a chromatographic method

using the Konig-reaction for final determination. The analysis of nicotin-
amide in biological material, such as tissues or body fluids, requires highly
selective separation methods to deal with a variety of parent structures and
metabolites. Usually, fluorometric or U.V. photometric determinations are
used for nicotinamide and the pyridine nucleotides.
For the assessment of vitamin activity- in biological material, the micro-
biological assay with lactohacilhis plantarum is suitable. The organism re-
sponds to nicotinamide and nicotinic acid. Nicotinic acid alone is assayed
with lenconostoe mesenteroides.
:
1 1 8 Nicotinic Acid and Nicotinamide
No International Units have been defined for this vitamin. Anahtical

results are expressed in terms of weight units of nicotinamide. The biological
vitamin activity of preparations can be assayed by the curative dog pellagra
test (black-tongue disease) or by the growth test in chicks or rats.
Pijjsiological Functions
Nicotinic acid, in the form of its amide, is the active group of the hydro-
gen-transferring coen2ymes nicotinamide adenine dinucleotide (NAD) and
nicotinamide adenine dinucleotide phosphate (NADP). Nicotinic acid is
readily converted to nicotinamide in the body.

Hydrogen transfers catalysed by NAD
and NADP play decisive roles
in intermediary metabolism. On
hand these coenzymes, bound to
the one
specific apoenzymes, intervene in oxidations and reductions in the degrada-
tion and synthesis of fatty acids, carbohydrates and amino acids on the ;
other hand the coenzymes, again linked to specific apoenzymes, fulfil a very
important function in the oxidative final degradation of the substrates in
the citric acid cycle, by taking-up the hydrogen in the substrate combustion
and transferring it to the flavin enzymes of the respiratory chain (see Vit-
amin B2, p. 98).
The process of biological oxidation is coupled with a considerable gain
of energy, which the body can store in the form of adenosine triphosphate
(ATP). The hydrogen-transferring coenzymes possess the property, im-
portant for their function, of combining with many apoenzymes which act
quite specifically on the substrates capable of dehydrogenation, in this way
channelling the hydrogen of many substrates into the reactions of the re-
spiratory chain.
The body is also capable of endogenous formation of nicotinic acid
from tryptophan Under normal conditions, however, the amounts of
.
nicotinic acid so formed are inadequate to meet the total nicotinic acid
requirement. Dietary supply of the vitamin is therefore essential.
Symptoms of deficiency of this vitamin can be considered under three

headings
1. Skin changes (pellagra erythema in man).
2. Lesions of the mucous membranes of the mouth , tongue, s tomach
and intestinal tract.
3. Changes of nervous origin.

Nicotinic Acid and Nicotinamide 119
Nicotinic Acid and Citric Acid Cycle
Fatt)' acid Carbohydrate Amino acid

degradation degradation degradation
NAD
Respiratory Chain
;
In humans the skin changes are characteristic symptoms, known as
"Pellagra", meaning coarse skin. The symptoms appear especially in areas

exposed to sunlight such as the face, nape of the neck, upper side of hands
and forearms. Initially the lesions are similar to sunburn. In chronic cases
the affected parts become squamous and assume a dark colour. Fewer skin
changes are seen in live-stock.
Changes in the digestive tract are observable in man as well as in other
animals in the first stages of deficiency. Depending on the degree of de-
ficiency, the symptoms appear as loss of appetite, dizziness, vomiting,
constipation and diarrhoea. In severe deficiency in man the tongue and
gastric mucosa are inflamed, the tongue becoming bright red and swollen
(raspberry-tongue).
Nervous disorders appear as insomnia, fatigue, vertigo and headache.
Severe cases lead to depression and confused states.
General condition Reduced growth, l oss of appetite, p oor feed util-

and growth isation (pig, fox, dog, cat, rabbit, monkey, rat,
mouse, chicken, turkey).
Skin and mucous Inflammation and ulceration of the mucous mem-

membranes branes of the tongue, mouth and oesophagus;
dark blue pigmentation of the edges and tip of the
tongue (« black tongue disease ») (dog, fox);
Squamous dermatitis (ears and back: pig; chicken;
area of nose and mouth : rat)
Coarse hair-coat (pig);

Inflammation of the buccal cavity and the anterior
part of the oesophagus, dark red colouration of
the mucous membranes (chicks);
Sensitivity to sunburn (trout).
Nervous system Disordered reflexes, ataxia, epileptic attacks (dog);
Jerking swimming movements (trout).
Bones Perosis (usually without slipping of AchiUes ten-

don);
Deformation of the femur (chicken, turkey, duck).
Blood and vascular Normocytic anaemia (pig, fur-bearing animals).

system
Dermatitis in
poultry resulting
from nicotinic
acid deficiency.
Dermatitis in the
areas of the ears,
neck and back of the
pig, resulting from
nicotinic acid
deficiency.
Gastro-intestinal Lack of appetite, vomiting , fr equent haemorrhagic

tract diarrhoea (rat, rabbit, dog, pig, cat, fur-bearing
animals, chicks);
Necrotic inflammation of large intestine and
caecum (pig);
Gastric and intestinal oedema (trout).
Human Requirements
The requirement data published in the literature include the amounts

of nicotinic acid which can be formed in the body from the tryptophan
ingested with food. The utihsation of the tryptophan as a precursor of
nicotinic acid depends on the dietary content of other essential amino-acids
such as leucine, isoleucine, valine, threonine and lysine and on vitamins
such as Bi, B(, and biotin. If the supply of one of the mentioned essential
amino-acids falls, less tryptophan is utilised in protein synthesis and more
becomes available for conversion to nicotinic acid. Calculation of the
requirement is based on the equivalence between 60 mg tryptophan and
I mg nicotinic acid. The nicotinic acid requirement is closely related to
body weight and to calorie intake. The minimum amount of nicotinic acid
(including that formed from tryptophan) capable of preventing pellagra is
4.4 mg per 1000 cal., provided that there is an intake of at least 2000 cal.
Alternatively on the assumption that the requirement for nicotinic acid

is about ten times greater than for vitamin Bj, average values of 6-18 mg
per day are obtained, depending on age. Requirements are increased in
digestive disorders, feverish illnesses, periods of growth, hard physical
work, pregnancy and lactation and ingestion of large quantities of fluid.
R
Pantothenic Acid
Free pantothenic acid is an unstable, extremely hygroscopic oil; it is
therefore unsuitable for practical application and is used mainly in the form
of c alcium and sodium salts. Panthenol, the alcohol corresponding to
pantothenic acid, has also assumed great importance, possessing full panto-
thenic acid activity.
In nature pantothenic acid only occurs rarely in the free state; it is,
however, very widely distributed as a component of coenzyme A, and it

occurs, above all, in liver, kidney, muscle, brain and egg-yolk as well as
in yeast, cereals and so me green plants (especially legumes ').
Pantothenic acid is optically active; only the dextro-rotatory forms

(calcium D-pantothenate, sodium D-pantothenate, D-panthenol) have vit-
amin activity.
Structural formula CH2OH — CiCHalz — CHOH — CO — NH — — — CH2 CH2
Pantothenic acid: R=COOH

Panthenol: R=CH,OH
Empirical formula Calcium salt: (C9Hi605N)2Ca

Sodium CgHjÔjNNa
salt:
Panthenol: CgHjgO^N
Molecular weight Calcium salt: 476.5

Sodium salt: 241.2
Panthenol: 205.3
Description Calcium salt: White powder

Sodium salt: White powder
Panthenol: Colourless, viscous oil, may crystallise
in storage
Solubility Calcium salt: Readily soluble in water ( ca. 40g/
100 ml), soluble in glycerol, very sparingly soluble

in alcohol, insoluble in ether, acetone and chloro-
form.
Sodium salt: Very soluble in water, slightly in
alcohol, insoluble in ether, acetone and chloro-
form.
Panthenol Very soluble in water, readily soluble in
:
alcohol, slightly in chloroform, sparingly in ether.

Melting point Calcium salt: Decomposition at ca. 200 °C

Sodium salt: ca. 160-165 °C
Specific rotation [a]^ Calcium salt: -1-26.0° to +28.0°

(c=4 in water)
Sodium salt: +26.5° to + 28.5°
(c=4 in water)
Panthenol: +29.5° to + 31.5°
(c = 5 in water)
Stability In cold storage and protected from moisture,

calcium and sodium D-pantothenate and D-pan-
thenol are fairly stable to atmospheric oxygen and
light. All three compounds are hygroscopic, par-
ticularly the sodium salt. Aqueous solutions of
these pantothenic acid derivatives are thermolabile
and undergo hydrolytic cleavage, especially if acid
or alkaline. Aqueous solutions of D-panthenol,
especially on the acid side, are significantly more
stable.
Methods of Determination in Simple Media
The chemical assay methods for pantothenic acid involve hydrolysis,

or reductive splitting of the compound and determination of one of the
cleavage products - ^-alanine or pantoic acid (a,>'-dihydroxy-^, /3-dimethyl-
butyric acid).
The /3-alanine moiety may be reacted with ninhydrin to a pink derivative
or with i,2-naphthoquinone-4-sulfonate to a brownish-yeUow^ derivative.
Treatment with potassium permanganate and 2,4-dinitrophenyl hydrazine
yields a compound which dissolves in pyridine with a blue colour.
The pantoic acid moiety may be reacted as the lactone with 2,7-naph-
thalenediol to a greenish-yellow complex or with alkaUne hydroxylamine
and ferric chloride to a purple hydroxamate.
Alternatively, the hydrolysis products may be chlorinated and used to
Uberate an equivalent amount of iodine from iodide solution, the iodine
being measured colorimetrically.
The assays of panthenol are similar; treatment of the /3-alanol with
ninhydrin and n-butanol at alkaline pH yield a fluorescent derivative.
As the above-mentioned reactions are subject to interference from
various sources, the assay procedure includes chromatographic purifications,
mostly by ion exchange chromatography.
Pantothenic Acid 125
Gas chromatographic assays of the acetates of panthenol and pantothenic

and of the trifluoroacetate and trimethyl silyl derivatives are
acid ethyl ester
also available.
Pantothenic acid in complex mixtures (food, feedstuffs etc.) can only be

determined microbiologi cally. Prior to determination the pantothenic acid
must be liberated. There is no really standard procedure for this, so that
operations must differ from case to case; for example, extraction with water
or different solvent mixtures, with or without enzymatic treatment (papain,
diastase etc.). Since pantothenic acid is labile to bases and acids the analytical
material may not be extracted under alkaline or acid conditions.
The micro-organisms saccharomjces carlsbergensis and lactohacillus arab-
inosus are suitable for the assay of pantothenic acid. Pantoic acid can be
assayed with acetohacter suboxydans.
No International Units of the biological activity of pantothenic acid

have been defined. Analytical results are generally expressed in terms of
weight units of pantothenic acid, calcium pantothenate being used as the
standard substance; i mg pantothenic acid is equivalent to 1.08 7 mg cal-
cium pantothenate.
Pantothenic acid can be assayed biologically in the curative chick-growth
test.
Units no longer in use are the YeastGrowth Unit and the Chick Unit;
I Yeast Growth Unit is equivalent to 0.8 meg calcium pantothenate and
I Chick Unit to 14 meg pantothenic acid.
In the cell pantothenic acid is incorporated into coenzyme A (=coenzyme

of acetylation), which is the biologically active form of pantothenic acid.
Coenzyme A
(CoA) occupies a central position in intermediary metabolism,
being capable of forming energy-rich thioesters with carboxyHc acids and
so activating the otherwise weakly active acids. Among the different acyl-
CoA derivatives appearing in intermediary metabolism, acetyl-CoA ("ac-
The Central Position of Acetjl-CoA in Metabolism
Fatty acids Carbohydrates Amino acids
Steroids
(Cholesterol,
Steroid hormones)
COj.HzOand Energy
of
aminosugars
Acetylsulphonamides
Acctylhexosamines
.
tive acetate") is the most important, since t he degradation o£jat^ carbohy-

drates and an entire series of amino-acids c onverges on the co mmon stage
of acetyl-CoA. By directing the coenzyme A-bound acetate into the citric
acid cycle (see Nicotinic acid, p. 118) it is provided for oxidative final de-
gradation to carbon dioxide and water with a considerable gain of energy

(synthesis of adenosine triphosphate, ATP). On the other hand, acetyl-CoA
is the single starting material for many synthetic processes of which the
body is capable if adequately provided with pantothenic acid.
Acetyl-CoA makes available the active form o f acetate for the bio-
synthesis of long-chain fatty acids , p hosphatides, cholestero l, s teroid hor-
mone s and bile acids . Acetyl-CoA also has an important role in the transfer
of the acetyl group to certain acceptors, the formation of acetylcholine as
transmitter substance at the nerve endings having special significance. Acetyl-
CoA is also required for the acetylation of aminosugars which are compo-
nents of various mucopolysaccharides (hyaluronic acid of connective tissue,
mucopolysaccharides of cartilage and other skeletal substances). Finally,
acetyl group transfer plays an important role, to be regarded as detoxication,
in the acetylation of sulphonamides and related substances. Coenzyme A
also intervenes in the synthesis of the porphyrin skeleton of the blood
pigment by providing active succinate for the synthesis of (5-aminolae-
vulinic acid.
On the basis of studies in human volunteers and of observations on

pantothenic acid-deficient animals, de ficiency of pantothenic acid is shown
in the following symptoms:
1. Reduced growth or weight loss.

2. Lesions of the skin and its appendages.
3. Disorders of the nervous system (peripheral sensitivity disorder,
paraesthesia, burning feet-syndrome, feelings of heaviness in limbs).
4. Gastro-intestinal disturbances.
5 Inhibition of antibody formation.
6. Impairment of adrenal function.
The symptoms are of many forms and differ from one animal species
to another. A differential diagnosis to biotin deficiency is frequently diffi-
cult.
Since pantothenic acid is so very widely distributed in nature, clear

cut deficiency symptoms in man are rarely found in practice. Typical
pantothenic acid deficiency does, however, appear occasionally in animals.
Inflammatory
changes at the
corner of the beak,
at the eye-lids and
partly on the toes,
the typical appear-
ance of pantothenic
acid deficiency
disease in the
chicken.
; ;
I30 Pantothenic Acid
Faded and rough

plumage resulting
from pantothenic
acid deficiency in
poultry.
General condition Reduced growth, dec rease in appetite, poor feed

u tilisation (pig, dog, fox, rat, monkey, guinea-pig,
chicken).
Skin and mucous Depigmentation of hair or feathers (dog, silver

membranes of fox, rat, chicken);
the head Brown exudate round the eyes (pig, rat);
Loss of hair behind the ears and on the nape of

the neck, reddening of the skin, encrustation,
coarse hair-coat (pig, rat);
Scab-like crusts at the corner of beak, on the
eye-lids closed by exudate and occasionally on
the toes, rough feathering (chicken)
Swelling of gills, high mortality (trout).
Nervous system Irritability , c onvulsions, collapse and death (dog);

L ocomotor disturbances , sp astic gait of the hin d
legs ("goose stepping") (pig, dog);
Paralyses (fox)
Ataxia (pig, monkey).
Blood Normocytic anaemia (pig, rat).

;
Gastrointestinal tract Loss of appetite, haemorrhagic gastroen teritis,

colitis, i ntestinal ulceration and abscesses profuse
,
diarrhoea, ulcerating and necrotic glossitis (pig,

dog, rabbit, fox);
Fatty degeneration of liver ( fox, cat, chicken, dog).
Reproductive system I mpaired laving performance and hatchabilitv of

eggs (poultry);
Resorption sterility and failure of lactation (pig);
Sexual organs remain infantile (young sows)
High neo-natal mortality, absence of sucking re-
flex, muscular weakness, trembling, locomotor
disorder (piglet);
Disturbed embryonic development (principally car-
tilage and bone) (rat).
Adrenals Atrophy r educed lipoid content

, , hyperaemia,
haemorrhages in adrenal cortex (pig, silver fox).
Human Requirements
Because of the widespread occurrence of pantothenic acid in man's food,

human deficiency states are rarely observed. With a mixed diet of 2500 cal.
the average intake of pantothenic acid is 10 mg, and this appears to re-
present the daily requirement.
Biotin
The molecular structure of biotin contains three asymmetric carbon

atoms. Eight different stereoisomers are therefore possible, of which only the
dextro-rotatory, so-called d-Biotin occurs in nature and possesses vitamin
activity. The synonym vitamin H is also used.
Natural biotin occurs partly in the free state (vegetables, fruit, milk,
rice-bran) and partly in a form bound to protein (animal tissues, plant
seeds, yeast). Important biotin sources for human nutrition are liver, kidney,
meat, yeast, egg-yolk, milk, mushrooms and some vegetables.
Structual formula
HN NH
I
HC -CH
H2C
I I
CH — CH2 — CH2 — CH2 — CH2 COOH
Empirical formula C,oH,603N,S
Description White crystalline powder
Solubility Soluble in dilute alkali, very sparingly soluble in

water (ca. 20 mg/ioo ml) and alcohol, insoluble
in most organic solvents.
2
Melting point 228-23 °C (with decomposition)
Specific rotation [a]^°= +90° to +94° (c=i.o in 0.1 N NaOH)

Stability Dry, crystalline d-biotin is fairly stable to air,
daylight and heat; it is gradually destroyed by

ultraviolet radiation. Aqueous solutions are rel-
atively stable if weakly acid or weakly alkaUne.
In strongly acid and alkaline solutions the biolog-
ical activity is destroyed by heating.
Purified, high potency solutions of biotin may be assayed by a photo-

metric method based on the splitting of an avidin dye complex by biotin.
Biotin 153
The anionic dye, 4'-hydroxyazobenzene-2-carboxylic acid, when bound to

avidin has an absorption maximum at The dye may be displaced
500 nm.
from its binding site by biotin which is more strongly bound to avidin. A
decrease in absorbance at 00 nm results, which is proportional to the amount
5
of biotin added.
Alternatively, biotin may be oxidised with potassium iodate to the sul-
fone, the iodate being reduced to iodine. The latter may be extracted and
quantified by colorimetry. These assays are, however, not generally suitable,
since biotin usually occurs only in low concentration, even in media of
simple composition, so that microbiological methods must be used.
A procedure for the determination of biotin in pharmaceutical injectable
preparations using gas-liquid chromatography of biotin silyl ester has also
been described.
The microbiological assay is the method of choice for biotin assay.
Suitable organisms are neurospora crassa, allescheria boydii and lactohacillus
plantarnm. The biotin must be extracted in the free state from the analytical
sample before assay.
No International Units have been defined for the biological activity of

biotin. Analytical results are generally expressed in terms of weight units
'^ ,Sl,jl[2 oj\A.oJu<y^
*^^P"^^ d-biotin. Biotin can be assayed biologically by assessment of the
^Ho •
•
. r healing effect of a biotin-containing preparation on skin lesions induced in
)^jyifî£j,c^
'
\ r y rats by massive feeding of egg-white ; protem-

the latter contains a protein-like
- 1 constituent avidin,
, w hich combines specifically with biotin an d prevents its
^\/(/)/M + t5rc '
a bsorption, leading to a true biotin deficiency.
^;;(ijuASL£^
î^4«^ A unit no longer used in practice is the Rat Unit; i meg d-biotin is
equivalent in activity to 0.2 Rat Unit.

}jqJL^^<^^^^'-^^^
The function of biotin in intermediary metabolism is associated with a

series of carboxylation reactions. Biotin bound to enzyme protein is capable
of taking-up carbon dioxide with formation of a C02-biotin enzyme com-
plex ("active carbon dioxide") and transferring it to a suitable substrate,
regenerating the free biotin enzyme complex. Such carboxylation reactions
are involved in the degradation of amino acids (leucine and isoleucine) and
also in the carboxylation of acetyl-CoA (CoA=coenzyme A; see Pantothenic
malonyl-CoA. In the body, malonyl-CoA can be combined
acid, p. 125) to
with molecule of acetyl-CoA, forming the corresponding acetyl-
a further
malonyl-CoA which, after decarboxylation, reduction and elimination of
134 Biorin
water is converted to butyryl-CoA. This derivative is again linked to malonyl-

CoA and a stepwise repetition of the reactions, as described for the syn-
thesis of butyryl-CoA, finally leads to formation of the long-chain fatty-acids
(palmitic, stearic acids). The biotin-dependent carboxylation of ace tyl -CoA
is thus a key reaction for th e body in the synthesis of fatty acids.
Further, the reversible carboxylation of pyruva te to oxalacetate - a
connecting link in the citric acid cycle - is biotin-dependent.
Biotin is thus playing an important part in gluconeogene sis, by which
the body maintains normal blood glucose concentrations s tarting from fa t
and protein whenever the carbohydrate intake with the food becomes
, in-
sufficient.
In biotin-deficient animals, an impairement of the synthesis of certain

proteins (e.g. serum albumin) and the activity or synthesis of some enzymes
(e.g. amylase, malic enzyme) has been observed. Biotin is, however, not
directly involved in these syntheses. The effect of biotin deficiency on pro-
tein synthesis is due to inadequate formation of dicarboxylic acids (oxalace-
tic, succinic) leading to a reduced incorporation of amino acids into pro-
teins.
Biotin and Lipogeiusis
Carbohydrates Lipids Phospholipids
Amino acids
Degradation of fats
Biotin 135
young
Biotin deficiency appears spontaneously in the suckling infant and
child (seborrhoeic dermatitis) and man, the
also in various animals. In
deficiency is always manifested by skin changes accompanied by non-
specific symptoms such as fatigue, los s of appet ite, nausea, m uscular pain,
h yperaesthesia, paraesthesi a and a fall in haemoglobin leveL
The development of biotin deficiency is favoured by the ingestion of
biotin-binding proteins (e.g. avidin from raw egg-white; stravidin and
streptavidin from certain streptomyces), high environmental temperatures,
and measures interfering with the biosynthesis of biotin by the intestinal
bacteria (e.g. therapeutic administration of antibacterials ;
preponderance
of certain carbohydrates in the food; modern animal housing systems mini-
mising the access to their faeces). Biotin in some grains (wheat, barley, milo)
and animal protein sources (meat meal, poultry by-product meal) is only
partially available to chickens and turkeys.
Some diseases (e.g. Fatty Liver and Kidney Disease in broilers) are re-
sponsive to biotin, although not being basically true biotin deficiency states.
General condition Reduced growt h, r educed appetite a nd poor feed

conversion (rat, mouse, pig, chicken)
Biotin deficiency in
chicken: Dermatitis
with encrustation
and cracking at
the corner of the
beak th e eye-lids
, ,
comb and under

the balls and toes
of the feet.
Biotin
136
Biotin deficiency:
Perosis in turkeys.
Enlargement of the
hock with lateral
distortion ; the
tendon of the
gastrocnemius
muscle frequently
slips from the
medial condyle
(healthy control on
the right). Besides
biotin deficiency,
deficiencies of
choline,manganese
and other micro-
nutrients often
give rise to these
symptoms.
Biotin 137
Skin (inclusive hair, Rough hair coat (pig, dog, mink, fox) and feather-
feathers, hooves ing (poultry), depigmentation (dog, mink, fox)
and beaks) and loss of hair (alopecia) (pig, dog, mink, fox).
Dry, scaly skin (poultry, pig) dark colouration of

;
skin ('blue slime') (trout).

Encrustations (chick, poult, pig) and fissures (pig)
in the angle of the mouth and on the toes and foot
pads (poultry) and on head, extremities, partic-
ularly the coronet (seborrhoeic eczema) (pig).
Transverse cracking of the foot pad; cracks in
hooves (pigs). Deformation of the beak (chicken)
Mucous membranes Furry tongue (pig)
Blood Fragmentation of erythrocytes (fish)
Locomotorv system Perosis (turkev, chicken)

Paralysis (calf, dog)
Muscular atrophy ; ataxia (newly hatched chicks);
Spastic convulsions (fish)
Reproductive svstem Impaired hatchability; malformation of embryonic

skeleton (shortened and deformed lower leg and
wing bones, adhesions between third and fourth
toes, parrot beak) (turkey, chicken).
Mink wit h severe '^

loss of fur in biotin
deficiency.
lig Biotin
Human Requirements
There is considerable difficulty in fixing exact requirements since biotin

is synthesised by intestinal bacteria. Account must be taken of the fact that
if raw egg-white is ingested, the avidin contained in it can bind biotin in

sush a way that vitamin activity is lost. On a daily intake of 1 50-300 meg
d-biotin deficiency symptoms do not appear.
r'^^"*''^ 1
Folic Acid
The structures of compounds with folic acid activity always contain one
or more linked molecules of glutamic acid which are essential for their
biological activity. Commercially synthesised folic acid has only one glu-
tamic acid group; it is also known as pteroylglutamic acid.
Folic acid occurs in nature chiefly as a conjugate; its distribution in

minute concentrations is spread throughout virtually all living cells. Folic
acid conjugates are present in liver, kidney, muscle (ox, calf), milk, cheese,
da rk leaf vegetables (in the free state in spinach) , cauliflower, l egumes and
wheat-germ.
Structural formula
OH COOH
CHz NH
NH^-C ^ CO NH —CH I
CH,
H,N CH2
I
COOH
Empirical formula
Description Yellow to orange-yellow crystalline powder.
Solubility Readily soluble in dilute alkali, soluble in dilute

acid, sparingly soluble in water, insoluble in al-
cohol, acetone, ether and chloroform.
Melting point Darkens at 250 °C, followed by charring.
Specific rotation [a]D=ca. ^20° (c=o.5 in 0.1 N NaOH).
Absorption spectrum Folic acid shows a characteristic absorption spec-

trum, dependent on the pH of the solution. In
0.1 N NaOH the maxima are at 256, 283 and
365 nm.
Stability C rystalline folic acid is fairly stable to air and heat,
but is degraded by light and ultraviolet radiation.

Neutral solutions are relatively stable acids, alkalis,;
oxidising and reducing agents have a destructive

effect.
140 Folic Acid
Assaj in Simple Mixtures
In pure solution, folic acid may be determined by U.V. spectrophoto-

metry at the above-mentioned absorption maxima in alkaUne solution. In
pharmaceutical preparations, reliable assay methods by polarography are
available. The pteroylglutamic acid molecule may also be split by reduction
with zinc hydrochloric acid or by oxidation with alkaline permanganate.
The resulting p-aminobenzoic acid moiety is then diazotised and coupled
with N-i-naphthyl ethylene diamine to produce a pink dye for colorimetric
determination. This technique is the official U.S. P. procedure. Alternatively,
the 2-amino-4-oxypteridine-6-carboxyHc acid moiety obtained upon per-
manganate oxidation may be determined by fluorometry. Combinations of
the colorimetric assay with thin-layer chromatographic purification to
eliminate interferences from Bratton- Marshall positive impurities have also
been described.
Assay in Complex Mixtures
The determination of folic acid in plasma, serum or whole blood is
performed by a competitive binding assay with radioactively labelled folate

and purified /S-lactoglobulin as binder.
In foods and feedstuffs, the assay is usually made by microbiological
methods. Suitable microorganisms are lactohacillus casei, streptococcus fae-
calis or tetrahymena geleii. This assay is not highly specific, since, in most of
the tests, folic acid can be replaced by nutritionally inactive or less active
compounds.
No International Units for the biological activity of folic acid have been
defined. Analytical results are generally expressed in weight units of folic
acid. Folic acid can be assayed biologically in chick- or rat-growth tests.
Folic acid, in the form s, 6, 7, S-tetr ^hvrlrofn lic acid^ is indispen^bl e for
the transformations of the one-carbon (C ,-) compounds in metabolism.
C, -units can be formed either by degradation of purines and histi-
The
dine, bv oxidative cleavage of glycine, by activation of free formate, by
elimination of the /?-carbon atom of serine with formation of glycine, or
by activation of free formaldehyde. The important physiological function
Folic Acid 141
of tetrahydrofolic acid consists of binding the Cj-units to the vitamin mole-

culeand thus transforming them to "active formic acid" or "active formal-
dehyde" so that these are interconvertible by reduction or oxidation, and
transferable to appropriate acceptors. Tetrahydrofolic acid also participates
in the reduction of active formaldehyde to the methyl group and the transfer
of the latter. This important reaction sequence enables the body to prepare
the Cj-compounds for a series of biosynthetic reactions : incorporation into
purine bases and histidine; transfer of active formaldehyde to glycine
with formation of serine; formation of methionine, choline, thymine and
methylnicotinamide by methylation of the corresponding precursors.
The purine bases (a denine, guanine) as well as thymine are important
constituents of the nucleic acids. Folic acid deficiency therefore leads to
serious disturbance of the biosynthesis oF the nucleic acids essential for cell
formation and function. The anaemias observed in folic acid deficiency are
related to this. In folic acid deficiency the formimino-glutamate formed as

an intermediate in the degradation of histidine can no longer be transformed
completely into glutamate and formiminotetrahydrofolic acid, and is there-
fore excreted in the urine. This excretion is suitable as a biochemical
diagnosis of folic acid deficiency, appearing at an early stage of deficiency.
Folic acid and Metabolism of C-^-compounds

Glycine
W Serine Histidine
C,
Pool of
Ji.
Choli C, > C -compounds
I
C, Purines
Folic acid
C,
/
c,
1/ C,
Methionine
\Thymine
Creatine Nucleic acids

;
142 Folic Acid
Vitamin Bj, is also closely associated with the progress of the folic acid-
dependent reactions of intermediary metabolism (see Vitamin Bj2, p. 113).
There is reason to believe that vitamin B12 participates in the reciprocal
transformation of active formaldehyde and active formic acid by oxidation
or reduction through the enzyme hydroxymethyltetrahydrofolic acid dehy-
drogenase.
Folic acid deficiency occurs in man and domestic animals under certain
conditions. In addition to inadequate nutritional supply, interference with
the intestinal flora (e.g. from sulphonamides or antibiotics) and disordered
absorption can also lead to a deficiency. Advanced folic acid deficiency is
always shown in changes of the blood picture (megaloblastic anaemia in

man). Such conditions are particularly pronounced if there is coincident
deficiency of folic acid and vitamin B,,. Further symptoms of folic acid
deficiency are lesions of the mucous membranes of the buccal cavity, and
sastro-intestinal disturbances leading to diarrhoea.
General condition Retarded growth, diminished appetite (pig, fox.
and growth rat, mouse, guinea-pig, chicken, turkey).
Skin and mucous Rough plumage, dermatitis and feather depig-

membranes of the mentation (chicks and turkey-poults);
head Darkening of the skin, brittleness of the tail fin
(trout)
Loss of hair (mouse).
Nervous system Cervical paralysis (stretched neck, trembling of

drooping wings) (turkey-poult).
Bones Perosis (turkey-poult).
Blood and Macrocytic anaemia, leucopenia, thrombopenia

vascular system (chicken, rabbit, fox, mink, cat);
Normocytic anaemia (pig).
Digestive tract Haemorrhagic gastro-enteritis, fatty liver degen-

eration (mink);
White watery diarrhoea (chick).
;
Folic Acid 143
Reproductive system Disordered fertility and lactation (breeding sows)

Poor hatchabilitv, malformations of the beak, and
distortion of embryonic femur (chicken).
Human Requirements
Since the intestinal flora of man and mammals svnthesise folic acid,
\yXsiA^ \ >^ 0^ no deficiency symptoms are seen, even on a diet free of folic acid ; deter-
p^j^^ S /t^ Q^ S^ niiii^tion of the requirement is therefore difficult. On the basis of adminis-
'
'
t) , \
tration of therapeutically acitve folic acid in nutritionally induced mega-
Icrtt o o- o*^ loblastic anaemia, the daily requirement of adult humans has been estimated
0.5-1 mg. During pregnancy and lactation an increased requirement of
about 5 mg is to be expected.
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/,
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:
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•976 - 270 - 72 076 1201
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Vitamiii;lli||i|

The Properties of the Vitamins

Vitamins and Chemicals Department

7 The Physiological Importance of the Vitamins for Man and Animals

Part II: The Individual Vitamins

(Physico-chemical properties, methods of determination, biological

The Physiological Importance

dietary of their physiological functions, a regular intake of some forty different

they comprise the actual energy-yielding and body-building substances

essential dietary group, they are recognised by two characteristic properties

Unidentified In this connection, the so-called "unidentified growth factors" (U.G.F.)

example, in residues of alcohol fermentation, in fish-solubles, grass sap,

The physiological importance of the vitamins for man and animals

Vitamins: Active Substances and Commercial Forms

Vitamin Group Principal Principal

Vitamin A Vitamin A (-alcohol) Vitamin A-acetate

Provitamin A /3-Carotene /5-Carotene

Vitamin D Vitamin D^ Vitamin D2

Vitamin E (anti- a-Tocopherol d-a-Tocopherol

Vitamin K Vitamin Kj Vitamin K,

Vitamin C Ascorbic Acid Ascorbic Acid

Vitamin Bj Thiamine Thiamine hydrochloride

Vitamin B, Riboflavin Riboflavin

The physiological importance of the vitamins for man and animals

Vitamin Group Principal Principal

Vitamin B^ Pyridoxine Pyridoxine hydrochloride

Vitamin B,2 Cvanocobalamin Cyanocobalamin

Niacin Nicotinic acid Nicotinamide

Pantothenic acid Pantothenic acid Calcium-D-pantothenate

Biotin d-Biotin d-Biotin

Folic Acid Folic acid Folic Acid

The physiological importance of the vitamins for man and animals

In countries where diets are unbalanced and inadequate, or where there

Examples of the most common avitaminoses are

several, vitamins. Thev appear in the form of ill-defined symptoms such as

skin changes, reduced vitalitv, lowered resistance to infections, etc.

In addition to the hvpovitaminoses and avitaminoses, which are due to Anti-vitamins,

bacteriostatics (sulphonamides, and coccidiostats) and antibiotics which,

Vitamin Every individual is constantly exposed to changing influences and

or more, according to the concentrations of the vitamins. Some vitamins

To cover vitamin requirements fully it is therefore necessary to add a

In the simplest case hypovitaminoses can result from inadequate or Hypovitaminoses

In replacement therapy of hypovitaminoses the doses generally used Pharmaco-

Vitamin A is used in lowered resistance to infections, lesions of the skin Fat-soluble

The areas of application of vitamin C are determined mainly by its Water-soluble

States resulting from increased demand (increased metabolism resulting

from different expert committees which are in good general

Vitamin supplementation levels now adopted by the food industry are

Revitamimsation: Compensation for losses in processing, i.e., restoration

The idea of revitaminisation is that vitamins should be added to those Revitaminisation

Vitamin Losses in Flour

\' itamin B, V itamin B. Vitamin B, Niacin

100 g wholemeal contain

SO that an important part of the supply of vitamins would be jeopardised

Vitamin Additions in fngjkg Flour

Vitamin Bj \'itamin B. Niacin

USA* 6.4 4.0 52.9

England 2.4 - 16.0

Switzerland** 2.81 1.70 33-67

* The higher levels of the revision 1974 have been considered.

A similar situation exists in countries where rice is a staple food. Hardly

example, to or above a definite standard. In this way the general principle

The question of compensating for vitamin losses arises also in evers*