OCHRATOXIN A: A REVIEW

Ochratoxin A is a mycotoxin of current interest from a public health point of view; consequently, a considerable effort has been and is being expended to document human and animal exposure to this mycotoxin and the consequences of such exposure. This paper reviews the current status of knowledge with respect to ochratoxin A. INTRODUCTION In recent years the attention of most mycotoxinologists has been focused to a great extent on the continuing quest for better ways to control exposure to aflatoxins, resolution of the questions relating to aflatoxin's acute and chronic toxicity (i.e., carcinogenicity) for humans, and an ever-expanding study of the myriad metabolites of the Fusaria (especially trichothecenes). Study of the ochratoxins, except in some localized areas of the world (e.g., Denmark, Sweden), has generally been of much lower priority. This situation seems to be changing, as evidenced by the recent efforts of public health officials in various countries (Germany, Canada, United States) to develop estimates of human risk from exposure to ochratoxin'A. in developing such risk estimates seems to be the paucity of data on human exposure to this mycotoxin and comparative toxicology data for translating observations in animals to humans. It has always been somewhat of an enigma that human exposure to ochratoxin seems to be localized in some countries of northern Europe and the Balkans and may possibly be responsible for a serious human nephropathy, but is not generally found in other areas of the world. Nevertheless, the International Program on Chemical Safety/World Health Organization, in its latest review of environmental contaminants, has recently selected ochratoxin as one of the mycotoxins for which sufficient new information indicates a need for further study and evaluation with respect to human health potential. Furthermore, the Commission on Food Chemistry, IUPAC, has embarked on a worldwide effort to document the natural occurrence of ochratoxin. It is the purpose of this paper to summarize recent information on the occurrence, toxicology and public health significance of human exposure to ochratoxin A. The main difficulty CHEMISTRY AND METHODOLOGY Biosynthetically the ochratoxins are pentaketides, consisting of a dihydroisocoumarin coupled to 8-phenylalanine (ref. l), with the following properties: White, odorless, crystalline solid HeOH M;, 168-173'C (9O'C, benzene) aD -46.8' (2650 pmol/L, CHC1,) 282 nm (cmX 890) , Mol. Wt. 403.8 332 nm (emax 63,300) hx 214 nm (emx 37,200) , Fig. 1. X Structure of ochratoxin A. Only ochratoxin A, and very rarely its dechloro analog, ochratoxin B, have been found in foods and feeds, whereas in culture filtrates of the generating molds, the methyl and/or ethyl esters as well as the 4-hydroxy-derivative and the isocoumarin nucleus of ochratoxin B (ochratoxin a ) have been observed (ref. 2). These compounds are produced by a variety of fungi, with the more frigophilic Penicillia (e.g., Penicillium viridicatum, 2. verrucosum) being the primary producers in the cooler areas of the world (ref. 3). 1030 Ochratoxin A: A review ANALYTICAL METHODOLOGY 1031 Several excellent reviews of analytical methods for determining ochratoxin A in foods and feeds (refs. 4, 5) are available. In general the major steps in all of the methods are the same: lot sampling, test sample preparation, extraction, cleanup, determination, confirmation of identity. Because the ochratoxin in most commodities is heterogeneously distributed, a relatively large random primary sample must be taken from the lot. In the case of grain, a 5-10 lb. sample is usually taken. This primary sample is then ground fine enough to pass a U.S. No. 20 sieve and blended thoroughly, and a 50-g test portion is withdrawn. It has been found that ochratoxin A in grains tends to degrade after grinding as a result of enzyme activity; consequently the test portion must be analyzed (extracted) immediately after grinding. The cleanup step most frequently used involves extraction into a basic solution (NaHC03), followed by acidification and partition into chloroform. The method that has received the widest acceptance has been collaboratively studied by the Association of Official Analytical Chemists (AOAC) and subsequently adopted by that organization as an official method (ref. 6). The IUPAC also selected it as a recommended method. In one of the earliest multimycotoxin methods, and one that has been extensively used (refs. 7, 8); the chloroform extract is put directly onto a silica gel column and aflatoxin and zearlenone are eluted; finally ochratoxin is eluted with acetic acidbenzene and determined as the free acid by thin-layer chromatography (TLC) and visual estimation or by TLC-densitometry. In all methods using fluorescence, sensitivity can be greatly improved by exposure of the developed TLC plate to NH which converts ochratoxin A to its ammonium salt and in so doing shifts rhe absorption maximum from 3 3 0 to 370 nm and increases the fluorescence emission at 460 nm. An added advantage of this technique is that the salt (anion form) of ochratoxin A appears to be more stable on a TLC plate than the free acid. An excellent high-performance liquid chromatographic (HPLC) method (ref. 9) applicable to the parts-per-billion level has been developed. The method includes the silica gel column cleanup described above preceded by an acidbase partition. A minicolumn screening method (ref. 10) has also been developed. Most of the older methods are time-consuming and use considerable amounts of solvent. validated which traps the ochratoxin on a 0.5-g C,, column impregnated with NaHC03. This method has been evaluated in a joint AOAC-IUPAC collaborative study (ref. ll), and on the basis of the results, was recommended to the AOAC for official first action status. The method consists of extraction of the test portion (50 g) by blending 3 min with 25 mL of 0.1 M H3P04 + 250 mL of CHCl,, filtering, and extracting the filtrate with 3% NaHCO,. The ochratoxin is trapped on a methanol-NaHC03-C,, column and recovered from the column by elution with EtOAc-MeOH-HOAc. HPLC. To avoid these disadvantages, a method has been developed and Ochratoxin A in the extract is determined by Over the last 15 years a number of immunochemical methods have been developed. These are very sensitive, specific and rapid. Some of these methods have been incorporated into test kits in the last few years and are commercially available. As is the case with all methodologies for trace contaminants, a source of standard material is required. Commercial sources of standard materials are listed in Table 1. The standard material as purchased, and solutions prepared from it, must be characterized for concentration by UV absorption measurements and for chromatographic purity by HPLC or TLC. Solution concentrations of about 30 pg/mL are required for adequate UV response, and about 100 pg/spot is needed for a TLC purity check. The identity of ochratoxin A in tlpositivett est extracts is usually confirmed by formation of the methyl or ethyl ester derivatives (ref. 6), followed by rechromatography (TLC, HPLC) or gas chromatography-mass spectrometry. 1032 COMMISSION ON FOOD CHEMISTRY Commodity Table 1 commercial Sources of Ochrag r c s Incidence Id2Y.a ReferNo. ( % Positive) (ng/g) Makor Chemical Co. $9.OO/mg Aldrich Chemical Co. 12 . 2 5/mg Sigma Chemical Co. 2 2.2 5/mg Chemical Dynamics Corp. 13.20/mg CSIR, S. Africa 7.OO/mg Barley Barley Barley malt Corn Corn Corn Feed Feed Oats Sorghum Wheat ITable 2 Jncidence of Ochratox in A in U.S. G r b 164 127 138 283 293 300 102 395 28 32 848