Food Additives & Contaminants
ISSN: 0265-203X (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/tfac19
A survey of cereals, cereal products, feedstuffs and porcine kidneys for ochratoxin a by radioimmunoassay Ladislav Fukal To cite this article: Ladislav Fukal (1990) A survey of cereals, cereal products, feedstuffs and porcine kidneys for ochratoxin a by radioimmunoassay, Food Additives & Contaminants, 7:2, 253-258, DOI: 10.1080/02652039009373889 To link to this article: http://dx.doi.org/10.1080/02652039009373889
Published online: 10 Jan 2009.
Submit your article to this journal
Article views: 7
View related articles
Citing articles: 3 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=tfac20 Download by: [University of Cambridge]
Date: 13 June 2016, At: 22:39
FOOD ADDITIVES AND CONTAMINANTS, 1990, VOL. 7, NO. 2, 2 5 3 - 2 5 8
A survey of cereals, cereal products, feedstuffs and porcine kidneys for ochratoxin A by radioimmunoassay LADISLAV FUKAL† Department of Biological Sciences, University of Agriculture, 165 21 Prague 6, Czechoslovakia
Downloaded by [University of Cambridge] at 22:39 13 June 2016
(Received 25 August 1988; revised November 1988; accepted 17 February 1989) A commercial kit for the radioimmunochemical determination of ochratoxin A has been validated for application to various foods and feedstuffs. Using this kit a survey was carried out for the occurrence of ochratoxin A in Czechoslovak cereals, cereal products, feedstuffs and pig kidneys. The results show that the most contaminated are feedstuffs and cereals, in 26.7% and 8.8% of the samples of which, respectively, the ochratoxin A level exceeded 20 µg/kg. None of the cereal products and 4.2% of the pig kidneys unfit for human consumption by visual examination possessed ochratoxin A concentration higher than 5 µg/kg. Of all the samples analysed, 57% contained no detectable toxin (less than 1 µg/kg). Keywords: ochratoxin A, radioimmunoassay, cereals, feedstuffs, pig kidney
Introduction Ochratoxin A, a naturally occurring mycotoxin, is produced by several species of Aspergillus and Pénicillium fungi, which are frequently present in cereals and feeds. Experimentally, feeding of ochratoxin diets has a deleterious effect on a number of animal species. Clinical syndromes associated with ochratoxin A include nephrotoxicosis, hepatoxicosis, teratogenesis and impaired immunological function. Ochratoxin A has been linked to porcine nephropathy and is a suspected factor in endemic Balkan nephropathy of man (Krogh et al. 1973, 1977). Human exposure to ochratoxin A can occur directly by consumption of contaminated plant food or indirectly by consumption of tissues of animals exposed to contaminated materials. Appropriate regular surveillance should indicate the action required, if any, to minimize human exposure. Conventional procedures such as thin-layer and high-performance liquid chromatography are expensive and time consuming for regular monitoring of ochratoxin A in foods and feeds. Immunochemical methods for the determination of ochratoxin A are sensitive and specific and allow the rapid analysis of a large number of samples (Pestka et al. 1981; Morgan et al. 1983, 1986; Lee and Chu 1984; Rousseau et al. 1985,1987). This is important for epidemiological purposes. The purpose of this work was to carry out routine monitoring of ochratoxin A in foods and feeds using a commercial immunoassay kit. †Present address: Department of Biochemistry, Institute of Chemical Technology, Suchbatar Street 5, 166 28 Prague 6, Czechoslovakia. 0265-203X/90 $3.00 ©1990 Taylor & Francis Ltd.
254
L. Fukal
Downloaded by [University of Cambridge] at 22:39 13 June 2016
Experimental
Reagents Ochratoxin A was supplied by Sigma Chemical (St. Louis, MO, USA). Kits for radioimmunoassay of ochratoxin A were obtained from the Institute of Nuclear Research (Kosice, Czechoslovakia). Sep-Pak d s cartridges were obtained from Waters Associates (Milford, MA, USA). The other chemicals used were from Lachema (Brno, Czechoslovakia). The radioimmunoassay (RIA) kits contain lyophilized standards of ochratoxin A, lyophilized rabbit antiserum against conjugate of bovine serum albumin with ochratoxin A, radioligand [125I]ochratoxin prepared by labelling of ochratoxin B with 125I, 0-1 M veronal buffer (pH 8-6), a 25% solution of polyethylene glycol and two cereal control samples containing 15 and 50/ig/kg of ochratoxin A. The antiserum showed cross-reactions of 100, 4, 27, 0, 0, 0 and 0'4% for ochratoxin A, ochratoxin B, ochratoxin C, L-phenylalanine, D-phenylalanine, aflatoxin Bi and citrinin, respectively. Standard solutions of ochratoxin A were prepared in veronal buffer containing 10% of acetone to achieve a concentration range of 5-320 pg per 100 \A. Calibration graphs were constructed for seven concentrations within this range. The lyophilized radioligand and lyophilized diluted antiserum were dissolved in veronal buffer. The radioligand solution and antiserum solution contained 7-3% and 0-1% of bovine 7-globulin, respectively. Extraction of samples Samples of cereals were obtained from silos, samples of cereal products from shops and samples of feeds from agricultural farms. Pig kidneys exhibiting macroscopic changes were collected from a slaughterhouse and stored at - 2 0 ° C until analysed for ochratoxin A. Ground cereal samples (2g) were extracted with a 0-5% solution of H3PO4 in chloroform. Porcine kidney homogenates (2g) were extracted according to the method of Rousseau et al. (1987) using a 0-5% solution of phosphoric acid in chloroform and rapid clean-up with a Sep-Pak Ci8 cartridge. The extracts were evaporated to dryness and dissolved in 4 ml of acetone. Aliquots of 100 /A of the acetone extract were added to 900 /il veronal buffer. Radioimmunoassay procedure Polystyrene test-tubes of 5 ml volume (Koh-i-noor, Hardtmuth, Dalecin, Czechoslovakia) were used for RIA incubations and measurements. Volumes of 100 /tl of standard solution or of a sample extract in veronal buffer, 100/il of radioligand (about 17000 cpm) and 100 /*1 of diluted antiserum were pipetted into test-tubes and the reaction mixture was incubated at 45 °C for 1 h. The mixture was then cooled (20 min, 4°C) and 0-5 ml of 25% polyethylene glycol solution, chilled to 4 °C, was added. The precipitate formed was removed by centrifugation at 2000 g for 30 min at 4 °C. The radioactivity of the precipitate free of the supernatant was counted for 60s on an NA 3601 7-counter (Tesla, Libérée, Czechoslovakia). The ochratoxin A content was calculated from calibration graphs of log (ochratoxin A concentration) vs. the ratio B/Bo (see figure 1) (Fukal et al. 1987). Application of the assay to the determination of ochratoxin A in samples was preceded by validation of the absence of matrix effects in the RIA used, with the
Ochratoxin A in foods and feedstuff s
255
Downloaded by [University of Cambridge] at 22:39 13 June 2016
working detection limit mentioned below. Agreement between this type of RIA and TLC in the quantification of ochratoxin A was verified by Ruprich et al. (1988). Results and discussion A typical calibration graph for the RIA determination of ochratoxin A is shown in figure 1. A linear relationship was found between log (concentration) and per cent bound in the range 20-160 pg per 0-1 ml, viz. log concentration = -0-020245/ Bo + 2-829 (correlation coefficient = 0-9998). For various lots of RIA kits, the average values of non-specifically bound (in the absence of antiserum) and zero specifically bound (in the absence of unlabelled ochratoxin A) varied in the ranges 7-8% and 47-52%, respectively. The sensitivity of the RIA was 5 pg per tube given by.two standard deviations from the zero value on the calibration graph and representing a detection limit of 1 /ig/kg. The intra- and inter-assay coefficients of variation for the ochratoxin A standard (20 pg per 0-1 ml) were 4-7% and 7-6%, respectively (n = 7). The effectiveness of the extraction and clean-up procedure is an important factor in determining the sensitivity of RIA for the determination of ochratoxin A in biological samples. Results for the recovery of ochratoxin A spiked in various materials are given in table 1. On the basis of these results, the RIA procedure was assessed as having a mean recovery value of 80% for samples of plant origin and 75% for kidneys. All survey results were corrected accordingly. A summary of the survey results for cereals, cereal products, feedstuffs and porcine kidneys is given in table 2. The Czechoslovak Hygienic Service has declared tolerance levels of 1, 5 and 20 /tg/kg for ochratoxin A in infant formulae, children's
100 B/Bo (%)
50-
0
5
10
20
40 80 160 320 OCHRATOXIN (pg/0.1ml)
Figure 1. Calibration graph for radioimmunoassay of ochratoxin A. B = bound radioligand in the presence of a known concentration of unlabelled ochratoxin; Bo = bound radioligand in the absence of unlabelled ochratoxin. The equation of linear regression was found to be log (concentration) = -0-0228 BjBo + 2-944; correlation coefficient = 0-9965.
256
L. Fukal Table 1. Recoveries of ochratoxin A from spiked samples using RIA.
Sample
Ochratoxin A added Oig/kg)
Recovery (%)
10 10 5
77-6 ± 6-1 81-4 + 5-8 78-2 ± 7 - 0
5 20 20 5
82-3 82-1 80-8 74-8
Downloaded by [University of Cambridge] at 22:39 13 June 2016
Wheat Barley Oat flakes Instant grain-based child food Poultry feedstuffs Pig feedstuffs Porcine kidney
±8-6 ±5-2 ±5-7 + 9-2
foods and other foods, respectively (Anon. 1986). None of the cereal products analysed exceeded the limit of 5 jig/kg. Only 4-2% of the pig kidneys possessed ochratoxin A concentrations higher than this limit and 70*4% contained less then 1 jig/kg. The most contaminated samples were found to be cereals and feedstuffs. Ochratoxin contents higher than 20/tg/kg were found in 8-8% of cereal samples (26,31,37 and 38 /tg/kg) and in 26-7% of feedstuffs (the highest levels being 107, 145 and 164 jig/kg). Many papers have given an indication of the extent of ochratoxin contamination in a wide variety of cereals and feedstuffs in various countries (e.g. Krogh et al. 1973, 1974; Shotwell et al. 1976; Abramson et al. 1983; Buckle 1983; Bauer and Gareis 1987). The proportion of samples in which the ochratoxin level exceeded 20jtg/kg ranged from 1 to 58%. In comparison with our results, some workers found higher proportions of contaminated pig kidneys exhibiting macroscopic lesions. A survey in Denmark indicated that 35% of kidneys of pigs with nephropathy (60 samples examined) contained ochratoxin A with a maximum Table 2. Results of a screening control of foods and feedstuffs for the content of ochratoxin A by radioimmunoassay.
Sample type Wheat Barley Wheat flour Oat flakes Instant grain-based child food Poultry feedstuffs Pig feedstuffs Pig kidneys with macroscopic lesions Total
Number of samples
Number of samples containing ochratoxin A in the range (pg/kg) 20
Downloaded by [University of Cambridge] at 22:39 13 June 2016
Ochratoxin A in foods and feedstuff s
257
concentration of 68 fig/kg (Krogh 1977). Therefore, a control programme was established to reduce the amount of ochratoxin A consumed via pork products. Porcine kidneys displaying macroscopic lesions are analysed for ochratoxin A and the entire carcass is condemned if the ochratoxin A concentration exceeds 25 ftg/kg. In 1983, of 7639 kidneys analysed, 29% contained more ochratoxin A than 25 /*g/kg (Büchmann and Hald 1985). In Sweden, Yugoslavia, Hungary and Poland, high levels of ochratoxin A were also found in porcine kidneys (Rutqvist et al. 1977; Pepeljnjak et al. 1982; Sandor et al. 1982; Golinski et al. 1985). Recently, immunoassays have been used for monitoring ochratoxin levels in pig kidneys. In the UK, Morgan et al. (1986) examined 303 pig kidneys unsuitable for human consumption for ochratoxin A by enzyme immunoassay. Of these, 79-8% contained less than 1 jtg/kg and only 2*7% contained more than 5 /tg/kg of ochratoxin A. Rousseau et al. (1987) subjected two negative and ten positive certified kidney samples of ochratoxin-contaminated pigs to radioimmunoassay. Concentrations of 4-3-108-2 ^g/kg were found in the positive samples. In Czechoslovakia, the first reported natural occurrence of ochratoxin A was in a fodder barley harvested in 1975, which contained 3800 /^g/kg (Veselâ et al. 1978). Piskac et al. (1979) described the finding of ochratoxin A in kidneys of 12 pig cadavers with nephropathy. In spite of the many advantages of using immunoassay for monitoring ochratoxin A contamination in food and animal feedstuffs, continuous monitoring has not been adopted. Finally, the regular control of ochratoxin A contamination using the commercial RIA kit is recommended for the Czechoslovak hygienic service. References ABRAMSON, D., MILLS, J. T. and BOYCOTT, B. R., 1983, Mycotoxins and mycoflora in animal feedstuffs in Western Canada. Canadian Journal of Comparative Medicine, 47, 23-26. ANON., 1986, Hygienic Regulations, Vol. 61, No. 69. (Prague: Avicenum), p . 21. BAUER, J. and GAREIS, M., 1987, Ochratoxin A in der Nahrungsmittelkette. Journal of Veterinary Medicine, Series B, 34, 613-627. BÜCHMANN, N. B. and HALD, B., 1985, Analysis, occurrence and control of ochratoxin A residues in Danish pig kidneys. Food Additives and Contaminants, 2, 193-199. BUCKLE, A. E., 1983, The occurrence of mycotoxins in cereals and animal feedstuffs. Veterinary Research Communications, 7, 171-186. FUKAL, L., PROŠEK, J., RAUCH, P., SOVA, Z. and KÁŠ, J. 1987, Selection of the separation step in the
radioimmunoassay for aflatoxin B1 using 1251 as a marker. Journal of Radioanalytical and Nuclear Chemistry, Articles, 109, 383-391. GOLINSKI, P., HULT, K., GRABARKIEWICZ-SZCZESNA, J., CHELKOWSKI, J. and SZEBIOTKO, K., 1985,
Spontaneous occurrence of ochratoxin A residues in porcine kidney and serum samples in Poland. Applied and Environmental Microbiology, 49, 1014-1015. KROGH, P. 1977, Ochratoxin A in residues in tissues of slaughter pigs with nephropathy. Nordisk Veterinaermedicin, 29, 402-405. KROCH, P., HALD, B. and PEDERSEN, H. E., 1973, Occurrence of ochratoxin A and citrinin in cereals associated with mycotoxin porcine nephropathy. Acta Pathologica et Microbiologica Scandinavica, Section B, 81, 689-695. KROGH, P., HALD, B., ENGLAND, L., RUTQVIST, L. and SWAHN, O., 1974, Contamination of Swedish
cereals with ochratoxin A. Acta Pathologica et Microbiologica Scandinavica, Section B, 82 301-302. KROGH, P., HALD, B., PLESTINA, R. and CEOVIC, S., 1977, Balkan (endemic) nephropathy and foodborne
ochratoxin A: preliminary results of a survey of fooodstuffs. Acta Pathologica et Microbiologica Scandinavica, Section B, 85, 238-240. LEE, S. C. and CHU, F. S., 1984, Enzyme-linked immunosorbent assay for ochratoxin A in wheat. Journal of the Association of Official Analytical Chemists, 67, 45-49.
258
Ochratoxin A in foods and feedstuff s
MORGAN, M. R. A., MCNERNEY, R. and CHAN, H. W. S., 1983, Enzyme-linked immunosorbent assay of ochratoxin A in barley. Journal of the Association of Official Analytical Chemists, 66, 1481-1484. MORGAN, M. R. A., MCNERNEY, R., CHAN, H. W. S. and ANDERSON, P. H., 1986, Ochratoxin A in pig
kidney determined by enzyme-linked immunosorbent assay (ELISA). Journal of the Science of Food and Agriculture, 37, 475-480. PEPELJNJAK, S., BLAZEVIC, N. and CULJAK; K., 1982, Histopathological changes and findings of ochratoxin A in organs of pigs, in the area of endemic nephropathy in Yugoslavia. In Proceedings of the Vth International IUPAC Symposium on Mycotoxins and Phytotoxins, Vienna, pp. 346-348. PESTKA, J. J., STEINERT, B. W. and CHU, F. S., 1981, Enzyme-linked immunosorbent assay for detection of ochratoxin A. Applied Environmental Microbiology, 41, 1472-1474. PISKAČ, A., HALOUZKA, R., DRÁBEK, J., MALÁ, J. and SMRČEK, Č., 1979, Mycotoxic nephropathy of pigs
in farming. Veterinářství, 29, 253-255.
Downloaded by [University of Cambridge] at 22:39 13 June 2016
ROUSSEAU, D. M., SLEGERS, G. A. and VAN PETEGHEM, C. H. 1985, Radioimmunoassay of ochratoxin A
in barley. Applied and Environmental Microbiology, 50, 529-531. RUPRICH, J., PISKAČ, A. and MALÁ, J., 1988, Using the radioimmunological assay of the ochratoxin A mycotoxin for the screening control of cereal-type foods. Veterinární Medicína (Prague), 33, 165-173. RUTQVIST, L., BJORKLUND, N. E., HULT, K. and GATENBECK, S., 1977, Spontaneous occurrence of
ochratoxin residues in kidneys of fattening pigs. Zentralblatt für Veterinärmedizin, Reihe A, 24, 402-408. SANDOR, G., GLAVITS, R., VADJA, L., VANYI, A. and KROGH, P., 1982, Epidemiologic study of ochratoxin
A-associated porcine nephropathy in Hungary. In Proceedings of the Vth International IUPAC Symposium on Mycotoxins and Phytotoxins, Vienna, pp. 349-352. SHOTWELL, O. L., GOULDEN, M. L. and HESSELTINE, C. W., 1976, Survey of U.S. wheat for ochratoxin and aflatoxin. Journal of the Association of Official Analytical Chemists, 59, 122-124. VESELÁ, D., VESELÝ, D., JELÍNEK, S. and KUSÁK, V., 1978, A finding of ochratoxin A in fodder barley. Veterinární Medicína (Prague), 23, 431-436.
ISSN: 0265-203X (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/tfac19
A survey of cereals, cereal products, feedstuffs and porcine kidneys for ochratoxin a by radioimmunoassay Ladislav Fukal To cite this article: Ladislav Fukal (1990) A survey of cereals, cereal products, feedstuffs and porcine kidneys for ochratoxin a by radioimmunoassay, Food Additives & Contaminants, 7:2, 253-258, DOI: 10.1080/02652039009373889 To link to this article: http://dx.doi.org/10.1080/02652039009373889
Published online: 10 Jan 2009.
Submit your article to this journal
Article views: 7
View related articles
Citing articles: 3 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=tfac20 Download by: [University of Cambridge]
Date: 13 June 2016, At: 22:39
FOOD ADDITIVES AND CONTAMINANTS, 1990, VOL. 7, NO. 2, 2 5 3 - 2 5 8
A survey of cereals, cereal products, feedstuffs and porcine kidneys for ochratoxin A by radioimmunoassay LADISLAV FUKAL† Department of Biological Sciences, University of Agriculture, 165 21 Prague 6, Czechoslovakia
Downloaded by [University of Cambridge] at 22:39 13 June 2016
(Received 25 August 1988; revised November 1988; accepted 17 February 1989) A commercial kit for the radioimmunochemical determination of ochratoxin A has been validated for application to various foods and feedstuffs. Using this kit a survey was carried out for the occurrence of ochratoxin A in Czechoslovak cereals, cereal products, feedstuffs and pig kidneys. The results show that the most contaminated are feedstuffs and cereals, in 26.7% and 8.8% of the samples of which, respectively, the ochratoxin A level exceeded 20 µg/kg. None of the cereal products and 4.2% of the pig kidneys unfit for human consumption by visual examination possessed ochratoxin A concentration higher than 5 µg/kg. Of all the samples analysed, 57% contained no detectable toxin (less than 1 µg/kg). Keywords: ochratoxin A, radioimmunoassay, cereals, feedstuffs, pig kidney
Introduction Ochratoxin A, a naturally occurring mycotoxin, is produced by several species of Aspergillus and Pénicillium fungi, which are frequently present in cereals and feeds. Experimentally, feeding of ochratoxin diets has a deleterious effect on a number of animal species. Clinical syndromes associated with ochratoxin A include nephrotoxicosis, hepatoxicosis, teratogenesis and impaired immunological function. Ochratoxin A has been linked to porcine nephropathy and is a suspected factor in endemic Balkan nephropathy of man (Krogh et al. 1973, 1977). Human exposure to ochratoxin A can occur directly by consumption of contaminated plant food or indirectly by consumption of tissues of animals exposed to contaminated materials. Appropriate regular surveillance should indicate the action required, if any, to minimize human exposure. Conventional procedures such as thin-layer and high-performance liquid chromatography are expensive and time consuming for regular monitoring of ochratoxin A in foods and feeds. Immunochemical methods for the determination of ochratoxin A are sensitive and specific and allow the rapid analysis of a large number of samples (Pestka et al. 1981; Morgan et al. 1983, 1986; Lee and Chu 1984; Rousseau et al. 1985,1987). This is important for epidemiological purposes. The purpose of this work was to carry out routine monitoring of ochratoxin A in foods and feeds using a commercial immunoassay kit. †Present address: Department of Biochemistry, Institute of Chemical Technology, Suchbatar Street 5, 166 28 Prague 6, Czechoslovakia. 0265-203X/90 $3.00 ©1990 Taylor & Francis Ltd.
254
L. Fukal
Downloaded by [University of Cambridge] at 22:39 13 June 2016
Experimental
Reagents Ochratoxin A was supplied by Sigma Chemical (St. Louis, MO, USA). Kits for radioimmunoassay of ochratoxin A were obtained from the Institute of Nuclear Research (Kosice, Czechoslovakia). Sep-Pak d s cartridges were obtained from Waters Associates (Milford, MA, USA). The other chemicals used were from Lachema (Brno, Czechoslovakia). The radioimmunoassay (RIA) kits contain lyophilized standards of ochratoxin A, lyophilized rabbit antiserum against conjugate of bovine serum albumin with ochratoxin A, radioligand [125I]ochratoxin prepared by labelling of ochratoxin B with 125I, 0-1 M veronal buffer (pH 8-6), a 25% solution of polyethylene glycol and two cereal control samples containing 15 and 50/ig/kg of ochratoxin A. The antiserum showed cross-reactions of 100, 4, 27, 0, 0, 0 and 0'4% for ochratoxin A, ochratoxin B, ochratoxin C, L-phenylalanine, D-phenylalanine, aflatoxin Bi and citrinin, respectively. Standard solutions of ochratoxin A were prepared in veronal buffer containing 10% of acetone to achieve a concentration range of 5-320 pg per 100 \A. Calibration graphs were constructed for seven concentrations within this range. The lyophilized radioligand and lyophilized diluted antiserum were dissolved in veronal buffer. The radioligand solution and antiserum solution contained 7-3% and 0-1% of bovine 7-globulin, respectively. Extraction of samples Samples of cereals were obtained from silos, samples of cereal products from shops and samples of feeds from agricultural farms. Pig kidneys exhibiting macroscopic changes were collected from a slaughterhouse and stored at - 2 0 ° C until analysed for ochratoxin A. Ground cereal samples (2g) were extracted with a 0-5% solution of H3PO4 in chloroform. Porcine kidney homogenates (2g) were extracted according to the method of Rousseau et al. (1987) using a 0-5% solution of phosphoric acid in chloroform and rapid clean-up with a Sep-Pak Ci8 cartridge. The extracts were evaporated to dryness and dissolved in 4 ml of acetone. Aliquots of 100 /A of the acetone extract were added to 900 /il veronal buffer. Radioimmunoassay procedure Polystyrene test-tubes of 5 ml volume (Koh-i-noor, Hardtmuth, Dalecin, Czechoslovakia) were used for RIA incubations and measurements. Volumes of 100 /tl of standard solution or of a sample extract in veronal buffer, 100/il of radioligand (about 17000 cpm) and 100 /*1 of diluted antiserum were pipetted into test-tubes and the reaction mixture was incubated at 45 °C for 1 h. The mixture was then cooled (20 min, 4°C) and 0-5 ml of 25% polyethylene glycol solution, chilled to 4 °C, was added. The precipitate formed was removed by centrifugation at 2000 g for 30 min at 4 °C. The radioactivity of the precipitate free of the supernatant was counted for 60s on an NA 3601 7-counter (Tesla, Libérée, Czechoslovakia). The ochratoxin A content was calculated from calibration graphs of log (ochratoxin A concentration) vs. the ratio B/Bo (see figure 1) (Fukal et al. 1987). Application of the assay to the determination of ochratoxin A in samples was preceded by validation of the absence of matrix effects in the RIA used, with the
Ochratoxin A in foods and feedstuff s
255
Downloaded by [University of Cambridge] at 22:39 13 June 2016
working detection limit mentioned below. Agreement between this type of RIA and TLC in the quantification of ochratoxin A was verified by Ruprich et al. (1988). Results and discussion A typical calibration graph for the RIA determination of ochratoxin A is shown in figure 1. A linear relationship was found between log (concentration) and per cent bound in the range 20-160 pg per 0-1 ml, viz. log concentration = -0-020245/ Bo + 2-829 (correlation coefficient = 0-9998). For various lots of RIA kits, the average values of non-specifically bound (in the absence of antiserum) and zero specifically bound (in the absence of unlabelled ochratoxin A) varied in the ranges 7-8% and 47-52%, respectively. The sensitivity of the RIA was 5 pg per tube given by.two standard deviations from the zero value on the calibration graph and representing a detection limit of 1 /ig/kg. The intra- and inter-assay coefficients of variation for the ochratoxin A standard (20 pg per 0-1 ml) were 4-7% and 7-6%, respectively (n = 7). The effectiveness of the extraction and clean-up procedure is an important factor in determining the sensitivity of RIA for the determination of ochratoxin A in biological samples. Results for the recovery of ochratoxin A spiked in various materials are given in table 1. On the basis of these results, the RIA procedure was assessed as having a mean recovery value of 80% for samples of plant origin and 75% for kidneys. All survey results were corrected accordingly. A summary of the survey results for cereals, cereal products, feedstuffs and porcine kidneys is given in table 2. The Czechoslovak Hygienic Service has declared tolerance levels of 1, 5 and 20 /tg/kg for ochratoxin A in infant formulae, children's
100 B/Bo (%)
50-
0
5
10
20
40 80 160 320 OCHRATOXIN (pg/0.1ml)
Figure 1. Calibration graph for radioimmunoassay of ochratoxin A. B = bound radioligand in the presence of a known concentration of unlabelled ochratoxin; Bo = bound radioligand in the absence of unlabelled ochratoxin. The equation of linear regression was found to be log (concentration) = -0-0228 BjBo + 2-944; correlation coefficient = 0-9965.
256
L. Fukal Table 1. Recoveries of ochratoxin A from spiked samples using RIA.
Sample
Ochratoxin A added Oig/kg)
Recovery (%)
10 10 5
77-6 ± 6-1 81-4 + 5-8 78-2 ± 7 - 0
5 20 20 5
82-3 82-1 80-8 74-8
Downloaded by [University of Cambridge] at 22:39 13 June 2016
Wheat Barley Oat flakes Instant grain-based child food Poultry feedstuffs Pig feedstuffs Porcine kidney
±8-6 ±5-2 ±5-7 + 9-2
foods and other foods, respectively (Anon. 1986). None of the cereal products analysed exceeded the limit of 5 jig/kg. Only 4-2% of the pig kidneys possessed ochratoxin A concentrations higher than this limit and 70*4% contained less then 1 jig/kg. The most contaminated samples were found to be cereals and feedstuffs. Ochratoxin contents higher than 20/tg/kg were found in 8-8% of cereal samples (26,31,37 and 38 /tg/kg) and in 26-7% of feedstuffs (the highest levels being 107, 145 and 164 jig/kg). Many papers have given an indication of the extent of ochratoxin contamination in a wide variety of cereals and feedstuffs in various countries (e.g. Krogh et al. 1973, 1974; Shotwell et al. 1976; Abramson et al. 1983; Buckle 1983; Bauer and Gareis 1987). The proportion of samples in which the ochratoxin level exceeded 20jtg/kg ranged from 1 to 58%. In comparison with our results, some workers found higher proportions of contaminated pig kidneys exhibiting macroscopic lesions. A survey in Denmark indicated that 35% of kidneys of pigs with nephropathy (60 samples examined) contained ochratoxin A with a maximum Table 2. Results of a screening control of foods and feedstuffs for the content of ochratoxin A by radioimmunoassay.
Sample type Wheat Barley Wheat flour Oat flakes Instant grain-based child food Poultry feedstuffs Pig feedstuffs Pig kidneys with macroscopic lesions Total
Number of samples
Number of samples containing ochratoxin A in the range (pg/kg) 20
Downloaded by [University of Cambridge] at 22:39 13 June 2016
Ochratoxin A in foods and feedstuff s
257
concentration of 68 fig/kg (Krogh 1977). Therefore, a control programme was established to reduce the amount of ochratoxin A consumed via pork products. Porcine kidneys displaying macroscopic lesions are analysed for ochratoxin A and the entire carcass is condemned if the ochratoxin A concentration exceeds 25 ftg/kg. In 1983, of 7639 kidneys analysed, 29% contained more ochratoxin A than 25 /*g/kg (Büchmann and Hald 1985). In Sweden, Yugoslavia, Hungary and Poland, high levels of ochratoxin A were also found in porcine kidneys (Rutqvist et al. 1977; Pepeljnjak et al. 1982; Sandor et al. 1982; Golinski et al. 1985). Recently, immunoassays have been used for monitoring ochratoxin levels in pig kidneys. In the UK, Morgan et al. (1986) examined 303 pig kidneys unsuitable for human consumption for ochratoxin A by enzyme immunoassay. Of these, 79-8% contained less than 1 jtg/kg and only 2*7% contained more than 5 /tg/kg of ochratoxin A. Rousseau et al. (1987) subjected two negative and ten positive certified kidney samples of ochratoxin-contaminated pigs to radioimmunoassay. Concentrations of 4-3-108-2 ^g/kg were found in the positive samples. In Czechoslovakia, the first reported natural occurrence of ochratoxin A was in a fodder barley harvested in 1975, which contained 3800 /^g/kg (Veselâ et al. 1978). Piskac et al. (1979) described the finding of ochratoxin A in kidneys of 12 pig cadavers with nephropathy. In spite of the many advantages of using immunoassay for monitoring ochratoxin A contamination in food and animal feedstuffs, continuous monitoring has not been adopted. Finally, the regular control of ochratoxin A contamination using the commercial RIA kit is recommended for the Czechoslovak hygienic service. References ABRAMSON, D., MILLS, J. T. and BOYCOTT, B. R., 1983, Mycotoxins and mycoflora in animal feedstuffs in Western Canada. Canadian Journal of Comparative Medicine, 47, 23-26. ANON., 1986, Hygienic Regulations, Vol. 61, No. 69. (Prague: Avicenum), p . 21. BAUER, J. and GAREIS, M., 1987, Ochratoxin A in der Nahrungsmittelkette. Journal of Veterinary Medicine, Series B, 34, 613-627. BÜCHMANN, N. B. and HALD, B., 1985, Analysis, occurrence and control of ochratoxin A residues in Danish pig kidneys. Food Additives and Contaminants, 2, 193-199. BUCKLE, A. E., 1983, The occurrence of mycotoxins in cereals and animal feedstuffs. Veterinary Research Communications, 7, 171-186. FUKAL, L., PROŠEK, J., RAUCH, P., SOVA, Z. and KÁŠ, J. 1987, Selection of the separation step in the
radioimmunoassay for aflatoxin B1 using 1251 as a marker. Journal of Radioanalytical and Nuclear Chemistry, Articles, 109, 383-391. GOLINSKI, P., HULT, K., GRABARKIEWICZ-SZCZESNA, J., CHELKOWSKI, J. and SZEBIOTKO, K., 1985,
Spontaneous occurrence of ochratoxin A residues in porcine kidney and serum samples in Poland. Applied and Environmental Microbiology, 49, 1014-1015. KROGH, P. 1977, Ochratoxin A in residues in tissues of slaughter pigs with nephropathy. Nordisk Veterinaermedicin, 29, 402-405. KROCH, P., HALD, B. and PEDERSEN, H. E., 1973, Occurrence of ochratoxin A and citrinin in cereals associated with mycotoxin porcine nephropathy. Acta Pathologica et Microbiologica Scandinavica, Section B, 81, 689-695. KROGH, P., HALD, B., ENGLAND, L., RUTQVIST, L. and SWAHN, O., 1974, Contamination of Swedish
cereals with ochratoxin A. Acta Pathologica et Microbiologica Scandinavica, Section B, 82 301-302. KROGH, P., HALD, B., PLESTINA, R. and CEOVIC, S., 1977, Balkan (endemic) nephropathy and foodborne
ochratoxin A: preliminary results of a survey of fooodstuffs. Acta Pathologica et Microbiologica Scandinavica, Section B, 85, 238-240. LEE, S. C. and CHU, F. S., 1984, Enzyme-linked immunosorbent assay for ochratoxin A in wheat. Journal of the Association of Official Analytical Chemists, 67, 45-49.
258
Ochratoxin A in foods and feedstuff s
MORGAN, M. R. A., MCNERNEY, R. and CHAN, H. W. S., 1983, Enzyme-linked immunosorbent assay of ochratoxin A in barley. Journal of the Association of Official Analytical Chemists, 66, 1481-1484. MORGAN, M. R. A., MCNERNEY, R., CHAN, H. W. S. and ANDERSON, P. H., 1986, Ochratoxin A in pig
kidney determined by enzyme-linked immunosorbent assay (ELISA). Journal of the Science of Food and Agriculture, 37, 475-480. PEPELJNJAK, S., BLAZEVIC, N. and CULJAK; K., 1982, Histopathological changes and findings of ochratoxin A in organs of pigs, in the area of endemic nephropathy in Yugoslavia. In Proceedings of the Vth International IUPAC Symposium on Mycotoxins and Phytotoxins, Vienna, pp. 346-348. PESTKA, J. J., STEINERT, B. W. and CHU, F. S., 1981, Enzyme-linked immunosorbent assay for detection of ochratoxin A. Applied Environmental Microbiology, 41, 1472-1474. PISKAČ, A., HALOUZKA, R., DRÁBEK, J., MALÁ, J. and SMRČEK, Č., 1979, Mycotoxic nephropathy of pigs
in farming. Veterinářství, 29, 253-255.
Downloaded by [University of Cambridge] at 22:39 13 June 2016
ROUSSEAU, D. M., SLEGERS, G. A. and VAN PETEGHEM, C. H. 1985, Radioimmunoassay of ochratoxin A
in barley. Applied and Environmental Microbiology, 50, 529-531. RUPRICH, J., PISKAČ, A. and MALÁ, J., 1988, Using the radioimmunological assay of the ochratoxin A mycotoxin for the screening control of cereal-type foods. Veterinární Medicína (Prague), 33, 165-173. RUTQVIST, L., BJORKLUND, N. E., HULT, K. and GATENBECK, S., 1977, Spontaneous occurrence of
ochratoxin residues in kidneys of fattening pigs. Zentralblatt für Veterinärmedizin, Reihe A, 24, 402-408. SANDOR, G., GLAVITS, R., VADJA, L., VANYI, A. and KROGH, P., 1982, Epidemiologic study of ochratoxin
A-associated porcine nephropathy in Hungary. In Proceedings of the Vth International IUPAC Symposium on Mycotoxins and Phytotoxins, Vienna, pp. 349-352. SHOTWELL, O. L., GOULDEN, M. L. and HESSELTINE, C. W., 1976, Survey of U.S. wheat for ochratoxin and aflatoxin. Journal of the Association of Official Analytical Chemists, 59, 122-124. VESELÁ, D., VESELÝ, D., JELÍNEK, S. and KUSÁK, V., 1978, A finding of ochratoxin A in fodder barley. Veterinární Medicína (Prague), 23, 431-436.
