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TECHNICAL REPORT
VITAMIN C (Ascorbic Acid)
INTRODUCTION :
Ascorbic Acid, or Vitamin C, is fundamental for maintaining the vital functions of animal organisms.
During the course of evolution, however, many species have lost their ability to synthesise it. This is
because the enzyme L-gulonolactone oxidase, responsible for a key function in the process, has
disappeared from their tissues (liver, kidneys).
These animals must, therefore, receive this enzyme in a pre-formed state in their feed.
A) CHEMISTRY AND PHYSICAL PROPERTIES
Chemically, Ascorbic Acid is 2 oxy-L-gulofuranolactone (enolic form).
MOLECULE FORMULA : C6H8O6 with a p.m. = 176.13
STRUCTURAL FORMULA

CHARACTERISTICS : a white, odourless, crystalline powder with the following physical
characteristics.
FUSION POINT : 190°-192°C (with decomposition)
DENSITY : 1.95
SOLUBILITY : 1 gr. in 3 ml. of water; in 30 ml. of ethyl alcohol, in 100ml of glycerol. Insoluble in
apolar organic solvents, in oils and in fats.
B) BIOCHEMICAL FUNCTIONS
Ascorbic Acid is essential for animal organisms because of its ability to :
1 – donate electrons;
2 – bind itself to substances and ions to form compounds.
Thanks to these two properties, Ascorbic Acid has a double role :
a) CATALYTIC : which manifests by keeping several enzyme systems active to the maximum, using a
mechanism which differs to that of the Vitamin B complex. b) PROTECTIVE : it can prevent, or at least attenuate, damage caused to the organism by toxic substances of an endogenous form or which come from the outside (feed, environment) and by pathogenic micro-organisms. TECHNICAL REPORT
1) FUNCTIONS TIED TO ITS ABILITY TO DONATE ELECTRONS :

Ascorbic Acid can donate 1 to 2 electrons at a time.
a) As a ‘2 electron donor’ : it is needed for the activity of the enzymes which catalyse the
R-H + Ascorbic Acid + O2 ---------------> R – OH + Dehydroascorbic Acid + H2O
Among these enzymes, those involved in synthesising collagen are particularly important, namely,
hydroxylase proline, procollagen – proline 2-oxiglutarate, 3-dioxygenase and hydroxylase lysine.
The most evident symptomatology of a lack of Vitamin C (lesions to the bone, cartilaginous tissue and
skin, haemorrhaging) is the altered formation of this structural protein and the systems connected to it.
Vitamin C’s involvement with hydroxylase enzymes, which are employed in the synthesis of carnitine,
peptide neurohormones and noradrenalin, has been shown recently.
b) As a ‘one electron donor’, Vitamin C can deactivate forms which are highly reactive to oxygen (O2
H2O2 and OH (radical hydroxyl) normally form during the processes of cell oxidation, but so do in especially high quantities under particular conditions of stress. This is undoubtedly the most macroscopic aspect of the protective role performed by the vitamin. As is known, in fact, these compounds, commonly called free radicals because of their high reactivity, can interact with various biomolecules. This provokes structural damage to them, which influences the functions they have. The favourite target of free radicals are the cell membranes, which are particularly rich in polyunsaturated acids and cholesterol. Through the transfer of an electron, Vitamin C can neutralise the reactivity of the free radicals and block the peroxidase processes of fatty acids even when they have already been started. Vitamin C, therefore, plays an important role in maintaining the vital functions which take place in the membranes (transportation of ions and molecules, energy production) The anti-radical action of Vitamin C manifests indirectly, too. It positively influences the balance between the oxidant substances and the defence systems (enzymatic and non) present in the cells. Vitamin E is one of the non enzymatic vitamins and, due to its liposolubility, can be considered as the main natural anti-oxidant for protecting membranes. This is because, as a ‘one electron donor’, it can interrupt the processes of lipidic peroxidasing, triggered off by the free radicals : R* + Vit. E -----------> RH + Vit. E* RCOO* + Vit. E --------> RCOOH + Vit. E* (radical Vit. E) The radical derivative of Vitamin C which forms, is reconverted into a vitamin by ascorbic acid, through the transfer of an electron. The radical derivative of ascorbic acid is, in turn, regenerated by semi-hydro ascorbate reductasis NADPH – dependent, present in the tissues : Vit. E* The anti-oxidising action of Vitamin C also takes place in the phagocytosis cells which circulate in the blood. A considerable number of free radicals form in these cells (through a biochemical mechanism called ‘radical explosion’) and are used to kill the pathogenic germs removed from the blood circulation through phagocytosis. The large quantity of Ascorbic Acid in the phagocytic cells (10-100 times more than that in the blood) controls the production of radicals in order to conserve the integrity of these cells and the tissues they come into contact with. It does not, however, interfere with their ‘killer’ action. TECHNICAL REPORT
2) FUNCTIONS TIED TO THE ABILITY TO INTERACT WITH MOLECULES OR IONS

Vitamin C also has a protective role against endogenous or exogenous substances which may damage the
health of the animal organism.
Vitamin C has different functions here as well.
A) It facilitates the processes which catabolize these substances.
It has been shown that animals with an Ascorbic Acid deficiency are less able to reclean their organisms
of the substances introduced voluntarily by the environment or through feed (pollutants).
This detoxifying action :
a) improves the activity of the enzymes involved in these processes e.g. cytochrome P450 oxidase.
b) prevents the intermediates which form during these processes and fix onto the tissues.
B) It blocks the endogenous formation of toxic substances.
The ability Ascorbic Acid has to hinder the synthesis of nitrosamines (substances with a mutagenic
activity, and therefore cancerogenic) for nitrosation of amines or amides in the digestive system, is
particularly important.
HNO3 -------------> HNO2 + R-NH2 ----------> R-N-NO (of feed origin)
C) It influences the metabolism of the mineral elements.
Ascorbic Acid favours the absorption of certain mineral elements in the intestine.
This action may partly be due to its reducing properties (the same as iron which can only be absorbed in
its reduced form ‘oso’) and partly to its ability to chelate elements, giving place to a soluble compound
which transports faster through the enterocyte.
The formation of soluble chelates in the case of heavy metals (Lead, Cadmium, Mercury) means they are
removed more quickly and easily from the organism.
3) IMMUNITARY FUNCTION
The protective role of Vitamin C is excellent for the organism’s immune defences and helps keep the
organism’s immune system as efficient as possible.
3A) Non specific immune response
Ascorbic Acid uses various mechanisms to protect the organism against infectious agents:
a) as a cofactor of the hydroxylase reactions associated with the synthesis of collagen, it helps to keep
the natural barriers whole (skin and the coating materials of all apertures); b) it favours the production and secretion, through the immune cells, of immunomodulator substances
like interferon, complement component and C19. c) it controls the different aspects of neutrophil functions, namely :
- chemiotaxis, by intervening to regulate the enzyme 5-lipoxygenase involved in the synthesis of
leukotriene
- ‘killer’, as it protects the neutrophils and the tissues they come into contact with, from the oxidising
damage of the extracellular free radicals. TECHNICAL REPORT

3B) Specific immune responses
Ascorbic Acid intensifies both the immuno-cellullar-mediate and autoimmune responses, increases
lymphocyte proliferation and decreases the responses of hypersensitivity.
Ascorbic Acid also acts here indirectly, through the regeneration of Vitamin E which is able to increase
specific and non specific immune responses.
VITAMIN C IN FISH
Ascorbic Acid is an essential nutrient for many animals (primates, guinea pigs, birds, reptiles, insects) and most fish species. This is because it cannot be synthesised by the tissues due to a deficiency of the enzyme gulonolactone oxidase. For this reason, Vitamin C must be ingested in feed. Other ways of giving it, like single parenteral administration, direct admission in water in order to avoid the onset of a grave deficiency symptomatology with consequences which are often lethal, cannot be proposed. Many of the deficiency symptoms, even the earliest ones, are associated with an alteration in the collagen synthesis processes : - imperfect healing of wounds; - uncoordinated movement; - deformation of the spine (scoliosis, lordosis, kyphosis). Other symptoms are : - depigmentation of the tissues; - internal haemorrhaging; - anaemia. All this means animals weigh and grow less and that the feed is used inefficiently. A Vitamin C deficiency in fish negatively influences their reproductive performances, intervening on the quality of the gamete, in both males and females. How Vitamin C performs is still not completely clear at this level. It has been said, however, that this effect may be attributed, at least partly, to one of its implications in the hormonal metabolism. Ascorbic Acid is a cofactor of the synthesis of steroid hormones and neurohormones. This justifies the drop in estradiol levels in animals which have a deficiency of this vitamin. The presence of a large amount of Vitamin C in fish eggs, leads us to believe that the embryos have a role in the opening process and then during the endogenous nutrition phases. The relationships between Vitamin C, stress and reproduction are particularly interesting. The reproductive power of salmon decreases considerably when they are subjected to stress : - the plasmatic levels of cortisol increase: - the Vitamin C levels drop. The administration of high doses of Vitamin C renormalises the biochemical parameters and reproductive function. This last effect may mean the inhibition is removed, due to the excess of cortisol in the synthesis of the steroid hormones in the gonads. TECHNICAL REPORT
VITAMIN C REQUIREMENTS
The quantity of Vitamin C recommended by the NRC (1981) for salmon (this species has been studied more than the others) and fish in general varies from 50-100 mg per kg of feed. These values have been calculated during studies carried out by feeding trout and salmon with purified diets, integrated with various levels of Vitamin C. An increase in weight, the absence of deficiency pathology and specific biochemical indications like the oxyproline : proline ratio in collagen were the efficiency parameters . Today, there is the tendency to administer higher quantities of Vitamin C to fish than those recommended. This is done in order to strengthen its protective role even further, especially during periods of stress, often found in fish bred in tanks. This stress is due to the imperfect environmental conditions (temperature, concentration of O2, salinity of the water), nutritional imbalances, or after medical treatment. Larger amounts of Vitamin C are required for shellfish (shrimps : from 5 to 10 times), probably because it is more involved in the synthesis of collagen - the main constituent of their esoskeleton and of the substance which strengthens the cells. There are a number of contributing factors which make administering suitable amounts of Vitamin C to fish problematic. Firstly, it is very unstable as far as many chemical and physical agents are concerned. Once it has been added, first to the integrator and, through this, to the feed, this Vitamin tends to degrade quickly because of its interaction with other components : - trace elements (particularly, iron and copper, as the activate the oxidation reaction); - choline; - fats (particularly highly saturated fats as they can be oxidised easily and cause the production of Higher amounts of Vitamin C are lost during the work processes, especially: - during pelletizing; - during extrusion (where higher temperatures are reached than during pelletizing). The dampness and water used during work compromise the stability of the Vitamin. Another aspect which makes it difficult for a suitable amount of Vitamin C to be assumed by the fish is that it is highly soluble in water. This means that part of the Vitamin in the feed is lost the moment it is administered. THE VARIOUS FORMS OF VITAMIN C
In order to avoid or at least try to contain the loss of Vitamin C to acceptable values, the feed industry specialised in the production of feed for water-culture always tends to resort to new forms of Vitamin C. This is to ensure the Vitamin C level can be corrected using appropriate super-doses to ensure the fish have a sure supply of this important micro-nutrient. There are various types on sale : 1) sulphuric and phosphoric derivatives of Ascorbic Acid (ascorbyl-sulphate and ascorbyl-phosphate); 2) Ascorbic Acid added to particular substances and at various concentrations (silicone 4%) (vegetable 3) Micro-encapsulated Ascorbic Acid (protected with various kinds of film : derivatives of cellulose, TECHNICAL REPORT
ADVANTAGES OF USING THE NEW FORMS OF VITAMIN C
A) Ascorbyl-suphlate and ascorbyl mono or poly-phosphate :
By using these forms of Vitamin, its loss considerably reduced, both during preparation and when the
finished feed is being stored prior to use.
The problem of the Vitamin being transferred from the feed into the water, has not yet been resolved
satisfactorily as these new forms are also soluble in water.
Although this problem may be negligible in feed for those animal/fish species which eat from the surface
(the feed in this case, is consumed instantly), it may be grave for those species which eat from the tank
bed. The food is in contact with the water for a long time, meaning a large amount of the Vitamin in its
stable form is lost.
Another problem tied to the use of these forms of Vitamin is their availability.
Not all the ascorbyl derivatives are biologically available for all the species, Ascorbyl-2-sulphate, for
example, is only used by a very few species.
As far as ascorbyl-phosphates are concerned, their use strongly depends on the hydrolytic efficiency of
the enzymes present in the digestive apparatus.
We must also add their incredibly high cost, considering the limited amount of active principle.
2) Addition of Ascorbic Acid (silicon and vegetable oils)
The advantages of using Vitamin C in the forms created by adding the substances mentioned above to the
active principle are very modest in terms of stability and transfer into the water, as well as resistance to
the various work phases.
In fact, the level of protection is very low regards the active principle.
Moreover, in the specific case of silicon protection, there is a doubt about the successive bioavailability
of the Vitamin.
3) Protected Ascorbic Acid (micro-encapsulated) “Microvit C 500”
From the results of research carried out at the Vitaminology Centre in the University of Bologna, it
appears that the use of Vitamin C covered with saturated fatty acids (“Microvit C 500) considerably
reduces the losses which occur both during feed preparation and storage.
Thanks to the hydrophilic coating, much less of the active principle is lost in water.
Besides this, the bioavailability of micro-encapsulated Vitamin C is similar to that of crystalline Ascorbic
Acid. This is shown in the data relative to the Vitamin C content in the tissues (muscle and
hepatopancreas) of subjects fed with the two different forms.
Using micro-encapsulated Vitamin C, is more economically advantageous than using the ascorbyl
derivatives.
In fact, even by using more “Microvit C 500” to compensate the losses during the feed production phase
(it is more stable when administered in water), the cost of giving animals the micro-encapsulated Vitamin
C is much lower because much less Vitamin is used.

Source: http://www.iza-italia.it/pdf%20gb/VITAMINA%20C%20(RELAZIONE%20INGLESE)%20agg.pdf

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