FACT SHEET - www.micotoxinas.com.br

CITRININ

Leatherhead Food Research Association, Randalls Road, Leatherhead, Surrey KT22 7RY, England

Naturally occurrence

Citrinin was first isolated as a pure compound from a culture of Penicillium citrinum in 1931. Later, yellowish coloured rice imported from Thailand to Japan in 1951 was found to be contaminated with P. citrinin and subsequent investigations showed that isolates of the fungus produced citrinin. This was only part of the story as the ‘yellow rice problem’ had been recognised for many years to be associated with a number of different fungi and a range of mycotoxins that included P. citrinum and citrinin respectively.

citrinin.gif (4071 bytes)
Fig. 1 Structural formula of citrinin

Since then, a number of other species of Penicillium including P. verrucosum have been reported to produce citrinin. Because this fungus is the major producer of ochratoxin A in cereals such as wheat and barley, it is not surprising that both mycotoxins often occur together although citrinin is reported much less frequently.

However, this may not reflect the real situation as citrinin is often not tested for and when it is sought it can be partially lost during some analytical procedures. Other fungi cited as producing citrinin include Aspergillus terreus, A. carneus and A. niveus. Early studies had suggested citrinin to have potential as a powerful antibacterial agent but its potent effect on mammalian kidneys quickly ruled out this possibility.

Chemical and Physical Properties

Citrinin is a simple, low molecular weight compound that crystallises as lemon coloured needles melting at 172 Deg C. It is sparingly soluble in water but soluble in dilute sodium hydroxide, sodium carbonate or sodium acetate, in methanol, acetonitrile, ethanol and most other polar organic solvents. Some photodecomposition occurs in fluorescent light both in solution and in the solid state. It can be degraded in acid or alkaline solution, or by heat. Colour reactions include brown with ferric chloride, green with titanium chloride and deep wine-red with hydrogen peroxide followed by alkali. Mono-acetate, diethyl, methyl ester and dihydro derivatives can be prepared. Citrinin is capable of forming chelate complexes and is not particularly stable so that extraction protocols have to be carefully designed.

Toxicity and Importance

The LD50 of citrinin has been reported as about 50 mg/kg for oral administration to the rat 35-58 mg/kg (ip.) to the mouse and 19 mg/kg (ip.)to the rabbit. Citrinin causes kidney damage and mild liver damage in the form of fatty infiltration. Other effects include vasodilation, constriction of the bronchi and increased muscular tone.

It often co-occurs with ochratoxin A and has been implicated in mycotoxic nephropathy of pigs in Denmark, Sweden, Norway and Ireland and in avian nephropathies. Acutely lethal doses administered to rabbits, guinea pigs, rats, mice or pigs cause swelling of the kidneys with eventual necrosis. In mice, citrinin is embryocidal and fetotoxic while in rats citrinin induces similar effects and high doses are teratogenic. There is only limited evidence for the carcinogenicity of citrinin to experimental animals.

It has been suggested that citrinin may be implicated in the fatal human kidney disease, Balkan Endemic Nephropathy, along with other mycotoxins including ochratoxin A and further unidentified toxins. However, it would seem unlikely in normal circumstances that citrinin presents much risk to humans as it is unstable in cereal processing so that the greatest risk is probably to livestock, particularly pigs, feeding on contaminated cereal products.

Products affected and Natural Occurrence

Citrinin has mainly been found in rice, wheat, flour, barley, maize, rye, oats, peanuts and fruit and may co-occur in cereals with ochratoxin A. There is limited evidence of it surviving unchanged into cereal food products.

Sampling and Analysis

Cereal samples can be screened by TLC or ELISA while quantitative results can be provided by HPLC. Polar organic solvents such as acidified acetonitrile commonly including potassium chloride, acidified chloroform or methanol are used for extraction. A number of solvent systems are suitable for developing TLC plates but this needs care as streaking of the spots can occur with some solvents on silica plates. This problem is mostly avoided by using reverse-phase plates. Citrinin is visible on TLC plates under longwave UV light as a yellow fluorescent spot that changes to blue after spraying with boron trifluoride reagent. Spots representing as low as 1ppb of citrinin can be seen with clean cereal extracts. A few ELISA methods have been developed and are claimed to be sensitive enough to detect citrinin in agricultural products.

The success of HPLC methods depends on developing good clean up methods, taking precautions so that degradation does not occur during analysis and ensuring that the column and solvent system used provide efficient separation and good peak shape. Under optimum conditions concentrations of less than 10 ppb of citrinin can be detected in cereal extracts.

Stability and Persistence

There is little information of its fate during processing but it is degraded by heat and alkali. It decomposes at 175 Deg C and concurrently detoxifies at this temperature under anhydrous conditions. Under semi-moist conditions this can occur at a lower temperature of about 140 Deg C. Even though citrinin itself may be destroyed by heat, in certain circumstances toxic breakdown products have been demonstrated in the presence of water. A toxic compound designated as citrinin H1 formed by cyclisation of 2 citrinin molecules has been identified.

Citrinin is likely to be destroyed during brewing and studies have shown that over 90% is destroyed during germination of barley, with no citrinin surviving the mashing process of making wort from malt. Presence of propionic acid destroys citrinin when added as a preservative to protect stored barley destined for animal feed from moulding during storage.

Legislation and Control

There has been only limited toxicological evaluation of citrinin and there are currently no regulations or guidelines within the EC. Because of its co-occurrence with ochratoxin A, indirect control in some circumstances may coincidentally be provided within the EC by the statutory regulation of that mycotoxin.

Naturally occurrence

Citrinin was first isolated as a pure compound from a culture of Penicillium citrinum in 1931. Later, yellowish coloured rice imported from Thailand to Japan in 1951 was found to be contaminated with P. citrinin and subsequent investigations showed that isolates of the fungus produced citrinin. This was only part of the story as the ‘yellow rice problem’ had been recognised for many years to be associated with a number of different fungi and a range of mycotoxins that included P. citrinum and citrinin respectively. Since then, a number of other species of Penicillium including P. verrucosum have been reported to produce citrinin. Because this fungus is the major producer of ochratoxin A in cereals such as wheat and barley, it is not surprising that both mycotoxins often occur together although citrinin is reported much less frequently. However, this may not reflect the real situation as citrinin is often not tested for and when it is sought it can be partially lost during some analytical procedures. Other fungi cited as producing citrinin include Aspergillus terreus, A. carneus and A. niveus. Early studies had suggested citrinin to have potential as a powerful antibacterial agent but its potent effect on mammalian kidneys quickly ruled out this possibility.

Chemical and Physical Properties

Citrinin is a simple, low molecular weight compound that crystallises as lemon coloured needles melting at 172 Deg C. It is sparingly soluble in water but soluble in dilute sodium hydroxide, sodium carbonate or sodium acetate, in methanol, acetonitrile, ethanol and most other polar organic solvents. Some photodecomposition occurs in fluorescent light both in solution and in the solid state. It can be degraded in acid or alkaline solution, or by heat. Colour reactions include brown with ferric chloride, green with titanium chloride and deep wine-red with hydrogen peroxide followed by alkali. Mono-acetate, diethyl, methyl ester and dihydro derivatives can be prepared. Citrinin is capable of forming chelate complexes and is not particularly stable so that extraction protocols have to be carefully designed.

Toxicity and Importance

The LD50 of citrinin has been reported as about 50 mg/kg for oral administration to the rat 35-58 mg/kg (ip.) to the mouse and 19 mg/kg (ip.)to the rabbit. Citrinin causes kidney damage and mild liver damage in the form of fatty infiltration. Other effects include vasodilation, constriction of the bronchi and increased muscular tone. It often co-occurs with ochratoxin A and has been implicated in mycotoxic nephropathy of pigs in Denmark, Sweden, Norway and Ireland and in avian nephropathies. Acutely lethal doses administered to rabbits, guinea pigs, rats, mice or pigs cause swelling of the kidneys with eventual necrosis. In mice, citrinin is embryocidal and fetotoxic while in rats citrinin induces similar effects and high doses are teratogenic. There is only limited evidence for the carcinogenicity of citrinin to experimental animals.

It has been suggested that citrinin may be implicated in the fatal human kidney disease, Balkan Endemic Nephropathy, along with other mycotoxins including ochratoxin A and further unidentified toxins. However, it would seem unlikely in normal circumstances that citrinin presents much risk to humans as it is unstable in cereal processing so that the greatest risk is probably to livestock, particularly pigs, feeding on contaminated cereal products.

Products affected and Natural Occurrence

Citrinin has mainly been found in rice, wheat, flour, barley, maize, rye, oats, peanuts and fruit and may co-occur in cereals with ochratoxin A. There is limited evidence of it surviving unchanged into cereal food products.

Sampling and Analysis

Cereal samples can be screened by TLC or ELISA while quantitative results can be provided by HPLC. Polar organic solvents such as acidified acetonitrile commonly including potassium chloride, acidified chloroform or methanol are used for extraction. A number of solvent systems are suitable for developing TLC plates but this needs care as streaking of the spots can occur with some solvents on silica plates. This problem is mostly avoided by using reverse-phase plates. Citrinin is visible on TLC plates under longwave UV light as a yellow fluorescent spot that changes to blue after spraying with boron trifluoride reagent. Spots representing as low as 1ppb of citrinin can be seen with clean cereal extracts. A few ELISA methods have been developed and are claimed to be sensitive enough to detect citrinin in agricultural products.

The success of HPLC methods depends on developing good clean up methods, taking precautions so that degradation does not occur during analysis and ensuring that the column and solvent system used provide efficient separation and good peak shape. Under optimum conditions concentrations of less than 10 ppb of citrinin can be detected in cereal extracts.

Stability and Persistence

There is little information of its fate during processing but it is degraded by heat and alkali. It decomposes at 175 Deg C and concurrently detoxifies at this temperature under anhydrous conditions. Under semi-moist conditions this can occur at a lower temperature of about 140 Deg C. Even though citrinin itself may be destroyed by heat, in certain circumstances toxic breakdown products have been demonstrated in the presence of water. A toxic compound designated as citrinin H1 formed by cyclisation of 2 citrinin molecules has been identified.

Citrinin is likely to be destroyed during brewing and studies have shown that over 90% is destroyed during germination of barley, with no citrinin surviving the mashing process of making wort from malt. Presence of propionic acid destroys citrinin when added as a preservative to protect stored barley destined for animal feed from moulding during storage.

Legislation and Control

There has been only limited toxicological evaluation of citrinin and there are currently no regulations or guidelines within the EC. Because of its co-occurrence with ochratoxin A, indirect control in some circumstances may coincidentally be provided within the EC by the statutory regulation of that mycotoxin.