STABLE FORMS OF VITAMIN C:

ASCORBIC ACID AND STABLE ASCORBATE ESTERS

AS SOURCES OF VITAMIN C IN AQUACULTURE FEEDS

Tim O’Keefe

Aqua-Food Technologies, Inc.

Buhl, Idaho USA

Essentiality

Vitamin C is known to perform numerous biochemical and physiological functions

in both plant and animal metabolism (Tolbert, 1979). Most animals can synthesize this

vitamin in the form of ascorbic acid in amounts sufficient to prevent the clinical

symptoms of deficiency collectively known as scurvy. However, primates, guinea pigs,

fish, shrimp, and some insects, bats, and birds require a dietary source of vitamin C to

prevent or reverse scorbutic symptoms. Among these species, dietary essentiality of

vitamin C in fish and shrimp probably results from an absence or insufficiency of L-

gulonolactone oxidase (Wilson, 1973; Yamomoto et al., 1978). This enzyme is required

for biosynthesis of ascorbic acid from glucose or other simple precursors (Lehninger,

1971).

Function

Ascorbic acid is a strong reducing agent that provides electrons to functional

groups of other biochemicals and free radicals found in the aqueous phase of biologic

fluids. Two biochemical reactions commonly associated with the function of ascorbic acid

in animals are hydroxylation and reduction. There may also be other as yet undetermined

functions of ascorbic acid as suggested by Tolbert (1979).

Over the past 30 years a great deal of research has been conducted to study the

function of ascorbic acid in aquatic species. Effects of dietary ascorbic acid on growth,

morphogenesis, reproduction, and adaptation have been studied extensively in carp (Sato

et al., 1978), catfish (Lovell, 1973; Wilson and Poe, 1973; Mayer et al., 1978; Lovell and

Lim, 1978; Lim and Lovell, 1978; Li and Lovell, 1985; Lovell, 1982; Launer et al., 1978),

trout and salmon (Hilton et al., 1977a, 1977b, 1978 & 1979; Sandnes et al., 1984; Wahli

et al., 1977, 1985 & 1986; Grant et al., 1989), shrimp (Lightner et al., 1977; Magarelli et

al., 1979; Magarelli and Colvin, 1978; Lightner et al., 1979), tilapia (Jauncey et al., 1985;

Soliman et al., 1986) and even snake heads (Mahajan and Agrawal, 1980).

In all of these species the most studied, and perhaps best understood, function of

ascorbic acid is its role as a cofactor in hydroxylating lysine and proline of collagen. This

protein is the major component of connective tissue, including bone and cartilage.

Impaired collagen formation results in the classical vitamin C deficiency symptoms of

scurvy. These include lordosis and scoliosis, as well as poor growth, anorexia, reduced

wound healing efficiency, and hemorrhaging.

Tucker and Halver (1984) cited other evidence supporting a similar role of

ascorbic acid in hydroxylation reactions in carnitine synthesis. They suggested that early

vitamin C deficiency symptoms of lethargy and fatigue may be due to depleted muscle

carnitine. These symptoms have been reported in trout (Grant et al., 1989) and described

similarly as prolonged periods of torpor.

Ascorbic acid also serves as a cofactor in hydroxylation reactions involved in

excretion of drugs and toxicants. Its role in detoxification of organochlorine pesticides

was investigated by Wagstaff and Street (1971). It was shown that ascorbic activity was

required to perform specific detoxification reactions in the liver. Mayer et al. (1977) found

that exposure to the pesticide toxaphene resulted in reduced levels of whole body vitamin

C activity and decreased backbone collagen in fathead minnows and channel catfish. This

lead to the hypothesis that hydroxylation reactions may compete with one another for

available vitamin C activity, thereby increasing the requirement for ascorbic acid.

Studies with trout showed that ascorbic acid also plays a role in iron metabolism.

Hilton et al. (1978) reported increased iron levels in the spleens of scorbutic fish, along

with liver iron levels and hematocrit readings that were positively correlated to dietary

levels of supplemental ascorbic acid. These data lead to the conclusion that ascorbic acid

may control the release of iron within spleen tissue, thereby affecting the redistribution of

iron stores.

Aside from these biochemical

processes affecting growth and

morphogenesis of various species of fish,

vitamin C activity also has been linked

conclusively to reproduction as well as

adaptive responses such as disease

resistance. Sandnes et al. (1984) and

Soliman et al. (1986) showed that fish

transfer ascorbic acid to eggs just before

spawning, where it is used in larval

development. Other researchers have

observed increased resistance to bacterial infections by channel catfish (Lovell, 1982, and

Li and Lovell, 1985) and reduced mortality in trout caused by the protozoan

Ichthyophthirius multifiliis (Wahli et al., 1985, 1986 and 1995).

ffect of Dietary Vitamin C on Survival of Trout

Infected with Icthyophthirius

100

120

5 6 7 8 9 10 11 12 13 14 15

Days Post-infection

Cumulative Mortality (%)

AA 0

AA 50

AA 2000

Requirement

A fish’s requirements for vitamin C, like any other vitamin, are actually the

amounts of vitamin activity required per kg of body weight per day to achieve specific

physiological responses. At any response level, these requirements are affected by the size

of the fish and its physiological state, as well as

by nutrient interrelationships and environmental

factors (NRC).

Vitamin Concentration

In Tissue

Vitamin Activity in Feed

Optimum

Growth

Adaptive

Response

Vitamin Requirement

Vitamin Concentration

In Tissue

Vitamin Activity in Feed

Optimum

Growth

Adaptive

Response

Optimum

Growth

Adaptive

Response

Vitamin Requirement

In general, relatively low vitamin C

activity levels are sufficient for optimum growth

and feed conversion, which is usually the desired

response for fish that are cultured for food.

Maximal adaptive response, such as disease

resistance and tolerance to environmental stress,

require slightly higher levels. For maximum

tissue storage, as may be appropriate for

broodstock or smolting salmon, loading levels

would have to approach the point of excretion.

The nutritionist must take into account the desired response level and the whole body or

indicator tissue concentration associated with that response, and calculate a concentration

in the feed that will deliver that amount of vitamin C within the expected feeding range.

Different stability characteristics of various sources of vitamin C add to the

difficulty of providing the amount required. Careful consideration must be given to losses

of vitamin C activity that may be sustained during feed manufacturing and during the

storage time before feeding.

Stability

Crystalline L-ascorbic acid is reasonably stable when dry and in pure form.

However, it is easily oxidized in neutral or alkaline conditions. Oxidation by molecular

oxygen, transitional metals, or other oxidants is accelerated by moisture, heat, and light.

Ascorbic acid is first oxidized to dehydroascorbic

acid (DHA), which retains vitamin potency because it can

be recycled to ascorbic acid by specific reductases and

cofactors. However, in a second step DHA is rapidly and

irreversibly converted to 2,3-diketogulonic acid, which

has no vitamin C activity.

These oxidation steps occur quite rapidly in fish

feeds, especially in formulations with high water activity

(Putnam, 1976; Crawford et al., unpublished; and Grant, et

al., 1989). Economic losses are substantial in spite of manufacturing and storage methods

implemented within the feed manufacturing industry to reduce rapid loss of vitamin C

activity. Processing and storage methods that remove oxygen, reduce heat, and avoid

contact with iron, copper, and other metal salts measurably improve retention of vitamin C

activity in feed. However, truly effective ascorbic acid stability has been achieved only by

physically or chemically protecting it from oxidizing agents.

H-C-OH

Ascorbic Acid

One method of physically protecting ascorbic acid from oxidation is encapsulation.

Ethylcellulose, a water-soluble “coating” typically used as a compressible tableting

compound, has been somewhat effective in providing increased stability of pure

crystalline ascorbic acid during storage. However, when blended with other ingredients

and subjected to all of the processes involved in feed manufacturing, ascorbic acid with

this type of coating is only slightly protected. Hilton et al. (1977) reported manufacturing

losses of vitamin C activity ranging form 74 to 100% in feeds supplemented with

increasing levels of ethylcellulose-coated ascorbic acid. As with crystalline ascorbic acid,

they showed that absolute losses of vitamin C activity from the coated product increased

as supplement levels increased. However, when expressed as percent, the greatest losses

were in feeds with supplement levels below 320 ppm.

High melting point fats or waxes have also been used to protect supplemented

ascorbic acid, particularly in feeds for aquatic species. Feeding trials have shown that

these coating materials are highly digestible and do not adversely affect ascorbic acid

bioavailability. Fat and wax coated products have been demonstrated to be more effective

to varying degrees than ethylcellulose. Still, relatively high losses of vitamin C activity

occur if shear forces and heat disrupt the coating. This tends to limit the effective

application of these coated ascorbic acid products in extruded feeds.

As an alternative to encapsulation, several chemical methods of stabilizing

ascorbic acid (against oxidation) have been developed to maintain vitamin C activity in

aquaculture feeds. The most effective derivatives developed to date are 2-sulfate and 2-

phosphate esters. In these compounds esterification protects the 2,3-enediol of AA from

oxidation by substituting the 2-hydroxyl group with electron-dense phosphate or sulfate

groups.

L-ascorbyl-2-sulfate (AS) is perhaps the most

stable derivative of ascorbic acid yet discovered. It is

a natural metabolite of ascorbic acid, occurring in

the urine of primates, guinea pigs, and fish (Baker et

al., 1971). AS is not a biologically available source

of vitamin C in the guinea pig (Kuenzig, 1974) or

the rhesus monkey (Machlin et al., 1976), but at high

levels it has been shown to prevent vitamin C

deficiency symptoms in rainbow trout and channel

catfish (Halver, 1975). Based on growth data with

channel catfish, the bioavailability of vitamin C activity in AS has been estimated to be

almost one-fourth that of the of ascorbic acid on an equimolar basis (Murai et al., 1978).

O S

O -

H-C-OH

O -

O S

O -

H-C-OH

O -

H-C-OH

O -

L - ascorbyl - 2 - Sulfate

The most recently developed ascorbic acid derivatives to be used in aquaculture

feeds are phosphate esters. In 1987 Seib and Liao developed a process for producing a

mixture of mono-, di- and tri-phosphorylated esters of

ascorbic acid, called L-ascorbyl-2-polyphosphate

(APP). This product is presently sold by Hoffmann-

LaRoche under the brand name of Stay-C™. BASF

Corporation markets a similar mono-phosphate

(AMP) product as Aqua-Stab™. Both of these

esterified forms of ascorbic acid are extremely stable

under harsh feed manufacturing and storage

conditions. Like AS, the number 2-carbon in the

ascorbic acid molecule is chemically protected from

oxidation. Unlike AS, however, both APP and AMP have been proven to be equivalent to

ascorbic acid on an equimolar basis when fed to catfish (Brandt et al., 1985 and Robinson

et al., 1989), rainbow trout (Grant et al., 1989) and shrimp (Shigueno and Itoh, 1988).

H-C-OH

O -

O P

O -

H-C-OH

O -

H-C-OH

O -

O P

O -

L - ascorbyl - 2 - phosphate

Bioavailability

In attempting to protect any labile nutrient from destruction, the objective is to

stabilize the compound prior to consumption without compromising its biologic activity

within the animal. Bioavailability of a micronutrient such as vitamin C from a physically

altered or chemically derived form is evaluated on its ability to support growth, maintain

tissue levels, and support other physiologic functions relative to ascorbic acid.

Presently available coated or encapsulated ascorbic acid products, although not

very stable in many applications, have been shown to be good sources of vitamin C

activity (Hilton et al., 1977). Bioavailability of vitamin C from AS, most likely depends

on the presence of the enzyme sulfatase (Benitez and Halver, 1982) and the animal’s

ability to produce enough sulfatase activity to release required quantities of ascorbic acid

at all times. Likewise, APP and AMP require phosphatase activity to release their

protecting phosphate groups.

In studies designed to test bioavailability of vitamin C from AS, results seem to

vary with species and perhaps with environmental conditions. On the basis of growth and

tissue storage data, Halver et al. (1975) concluded that AS was equivalent to ascorbic acid

on an equimolar basis in meeting vitamin C requirements of rainbow trout. However,

catfish feeding trials showed that at levels below 200 ppm vitamin C activity from AS

growth was much lower than for fish fed 50 ppm of ascorbic acid (Murai et al. 1978).

Additionally, vitamin C activities in blood and liver tissues were higher in catfish fed

ascorbic acid than in those fed molar-equivalent AS. Sandnes et al. (1989) observed

similar differences in tissue levels of vitamin C activity in Atlantic salmon fed AS verses

those fed ascorbic acid. These data on fish, along with other studies documenting the

absence of anti-scorbutic effects in monkeys and guinea pigs raise serious questions about

AS as a useful source of vitamin C activity.

Documented anti-scorbutic effects in a wide range of animal species are cause to

consider phosphate esters of ascorbic acid to be the most potentially useful forms of stable

vitamin C. They have been shown to be active in guinea pigs (Imai et al., 1967, and

Machlin et al., 1979), the rhesus monkey (Machlin et al., 1979) shrimp (Shigueno and

Itoh, 1988), and several other aquatic species.

Data from tests conducted by Grant et al.

(1989) show equivalent growth of rainbow trout

fed casein-based purified diets initially containing

400 ppm vitamin C activity from ascorbic acid or

20 ppm vitamin C from APP. In the same

experiment, control fish fed no ascorbic acid

exhibited all of the classical symptoms of vitamin

C deficiency.

In another feeding trial with practical diets

containing either ascorbic acid or APP, rainbow

trout were grown for 252 days from first feeding

swim-up fry to market size. No statistically

significant difference in growth rates were

observed between those two groups, whereas fish fed the same diet without a

supplemental source of vitamin C exhibited scorbutic symptoms.

Trout Growth on Feeds

with AA or APP

0 20406080100110

Days on Feed

Average Fish Weight (g)

400 ppm AA

20 ppm APP

No C

Apparent equivalent activity was evident from ascorbic acid and AS tissue storage

data collected after feeding supplemental APP and ascorbic acid at various dietary levels

(Table 1). Whole body, total vitamin C activity levels in fish fed APP as the source of

vitamin C were approximately twice as high as those fed ascorbic acid. In fish fed graded

levels of vitamin C activity up to 100 ppm from APP, liver and kidney levels of vitamin C

activity appeared to increase up to the maximum amount fed. Maximum storage levels

were not determined.

To test reproductive performance of fish fed APP as a source of vitamin C, Grant

and coworkers raised fathead minnows through a complete egg-to-egg life cycle (Table2).

Reproductive success, as measured by number of eggs laid with subsequent normal larval

development, was statistically similar for fish fed ascorbic acid or APP.

Perhaps the most striking evidence of bioavailability of vitamin C activity from

APP is the adaptive response observed by Satyabudhy et al. (1989). Resistance to

infectious hematopoietic necrosis (IHN) virus in six-week-old trout was directly

proportional to APP feeding levels over the range of 20 to 320 ppm ascorbic acid-

equivalents. This dramatic response was observed in both vaccinated and non-vaccinated

test groups of fish, indicating an effect on both native and conferred immune response.

Summary Recommendations

Nutrition research has shown that vitamin C has biochemical functions that are

critical to life. Most animals can synthesize sufficient amounts of this vitamin in the form

of ascorbic acid. However, fish and shrimp are among the few other species that cannot.

They must consume adequate amounts of ascorbic acid almost daily.

The challenge in providing feeds supplemented with adequate levels of vitamin C

activity is complicated by the instability of ascorbic acid. Oxidation is accelerated in the

complex matrix of feed during manufacturing and storage. To address the need for more

stable sources of vitamin C, several effectively protected ascorbic acid products have been

developed. These include coated ascorbic acid as well as phosphorylated esters of ascorbic

acid. Each of these has advantages and disadvantages associated with their use. However,

all of them have been proven effective in providing essential vitamin C activity.

Based on the combination of experimental results and field experience, researchers

and vitamin companies recommend the following levels of vitamin C activity in feed at

the time that it is consumed:

Recommended Vitamin C Levels

Culture

Conditions

Vitamin C

(mg/kg Feed)

Feeding

250 - 500

Grow-out 75 - 125

Stress 150 - 300

Broodstock 500 +

Additionally, one vitamin company recommends a vitamin C level of 1000 mg per kg feed

whenever the immune system of fish is challenged, such as handling and grading,

vaccination, disease outbreaks and transfer of smolts to seawater. Their recommendation

is to feed this high level for 2 to 4 weeks before and at least 2 weeks following these

stressful events.

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