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"' 2 x. ‘1!“ Wrap” . ‘R .I N $5.3 INN III is? %_ “2‘“ A "III _. V.‘ ‘ . IIIINI 3‘: % 6%,. I . - I 53$} 1 4.33351 Vikkwgh‘ij " 2‘” ”3." C "r ‘L «7"73- QL 2%.:th k. If ‘V I 'II. ‘III 1333’: :5ng Q“: ".5333“: in; “% fi‘fi‘tw if: I32; 5. I. Rm 1% $31? 0.123% 333E; . '- ‘1"?- ‘III '1. . I E: l Wfi’: .11 ‘ I. ‘ ' I W f “IQ " ”WIPE-I g? :37": -' . WEE-3'91!" L I 1 «3.2%; mini! ‘4“ u 31.... filial": .I a! THESIS This is to certify that the thesis entitled The Effects of Withdrawal and Processing upon the Levels of Aflatoxins in the Tissues of Pigs fed a Contaminated Ration. presented by Romeu Mesquita Furtado has been accepted towards fulfillment of the requirements for Human Nutrition j. mfg/{W Major professor Date 11/03/80 0-7639 J“§h. ti III h :3"- : " :9.:! -ll” '”5 '1 ‘14! OVERDUE FINES: 25¢ per day per item RETURNING LIBRARY'MATERIALS; Place in book return to remove charge from Circulation records THE EFFECTS OF WITHDRAWAL AND PROCESSING UPON THE LEVELS OF AFLATOXINS IN THE TISSUES OF PIGS FED A CONTAMINATED RATION By Romeu.Mesquita Furtado A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science and Human Nutrition 1980 6- we 57/ 5’ ABSTRACT THE EFFECTS OF WITHDRAWAL AND PROCESSING UPON THE LEVELS OF AFLATOXINS IN THE TISSUES OF PIGS FED A CONTAMINATED RATION by Romeu Mesquita Furtado Two trials were conducted to determine the amount of time necessary for tissue clearance from pigs fed an afla- toxin contaminated diet. There were 20 pigs in each trial, with 4 being fed the control diet and 16 being used to deter- mine the time necessary for tissue clearance after removal from the contaminated diet. The spiked diets of trials 1 and 2 contained 551 and 355 ppb of aflatoxins B1 and B2, respec- tively. The feed of the control pigs in trial 1 was natural- ly contaminated with 20 and 31 ppb of aflatoxins B1 and B2, respectively, while in trial 2 the control feed was free of aflatoxins. The initial phase of each trial, in which the pigs were fed the control and spiked diets lasted for 42 days. In trial 1 there was rm) significant difference be- tween weight gains and organ weights of the control pigs and those fed the aflatoxin spiked diet. The control pigs on trial 1 gained 40 percent less with a 24 percent reduction in feed intake as compared to the controls in trial 2. Even the low levels of aflatoxins occurring naturally in the con- trol diet resulted in small amounts of aflatoxins in the liver and kidneys of the control pigs in trial 1. Romeu M. Furtado In trial 2, the basal diet was uncontaminated with aflatoxins, so the pigs fed the spiked diet had 30 percent heavier livers, gained 45 percent less weight and had a 32 percent reduction in feed intake. Aflatoxins were found widely distributed in all tissues of the pigs fed the spiked diet. The blood showed the lowest level of residual afla- toxins, followed by the spleen, muscle and heart in that order. The highest concentration of aflatoxins was present in the liver and kidneys. Except for these organs, the levels of the M1 and M2 metabolites were much lower than the parent aflatoxins. The mean percentage retention of afla- toxins was calculated to be 0.03 and 0.04 percent for B1 and 32, respectively. The withdrawal trial showed that one day after placing the pigs on an aflatoxin-free diet there was a significant decrease in the aflatoxin levels in all organs and tissues. Two days following withdrawal from the contaminated feed, tissues of only one pig contained trace amounts of aflatox- ins. Four days after placing the pigs on an aflatoxin-free diet, there were no detectable levels of aflatoxins in any of the tissues. Processing and cooking resulted in some reduction of the aflatoxins in the meat. The differences on comparing raw and processed tissues, however, were not statistically significant. Although cooking the fresh tissues had the greatest effect in reducing aflatoxin levels, it was still not very effective, with a mean maximum reduction of only Romeu M. Furtado 26 percent._ Thus, processing and cooking were not very effective in removing the residual aflatoxins. ACKNOWLEDGMENTS The author wishes to express his sincere appreciation to Dr. A.M. Pearson, for his help in setting up the project and his invaluable assistance during the completion of the study. Gratitude is also extended to Dr. L.R. Dugan, Jr. and Dr. I. Gray of the Department of Food Science and Human Nutrition and to Dr. S.D. Aust of the Department of Biochem- istry, as well as to Dr. E.R. Miller and Dr. M.G. Hogberg of the Department of Animal Husbandry, for serving on the Gui- dance Committee and for reviewing the thesis. Special thanks are expressed to Dr. E.R. Miller and Dr. M.G. Hogberg and also to the staff of the MSU Swine Farm for their coopera- tion and assistance in aiding the author during the experi- mental trial. The author gratefully acknowledges the support of the Departamento de Techologia de Alimentos da Universidade Fed- eral de Vicosa (Brazil), the Programa de Ensino Agricola Su- perior (PEAS, Brazil) and the Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES) for the opportunity and financial assistance that made it possible to carry out the course work and research reported herein. Special gratitude is also expressed to the author's wife, Marizinha, and daughter, Erica, for their devotion, encouragement, patience and understanding. ii TABLE OF CONTENTS LIST OF TABLES . LIST OF FIGURES. INTRODUCTION . REVIEW OF LITERATURE . Occurrence of Aflatoxins. Isolation of Aflatoxins Metabolism of Aflatoxins. Toxicity of Aflatoxins. Excretion and Tissue Distribution of Ingested Aflatoxins. Occurrence of Aflatoxins in Foods of Animal Origin. Stability of Aflatoxins in Foods. EXPERIMENTAL Feeding Trial Preparation of the Diet. Experimental Animals Slaughtering and Collection of Samples . . . . . Processing of Meat Tissues. Preparation for Processing Curing Procedure Ham Curing. Bacon Curing. iii Page vi ix I6 21 30 35 48 53 53 53 54 56 57 57 57 57 58 Page Smoking-Cooking Schedule , , , , . . 58 Cooking of Raw Hams, , . . . . . . . . . 59 Cooking of Cured Ham Samples . , . . . . 59 Frying Belly and Bacon Samples . , . . . 6O Broiling of Loin Samples . . . . . . . . 60 General Methods of Analysis for Aflatoxins . . . . . . . . . . . . . . . . 61 Sample Preparation for Extraction. . . . 61 Extraction of Aflatoxins from Tissues . . . . . . . . . . . . . . . . 61 Purification of the Aflatoxin Extract . . . . . . . . . . . . . . . . 62 Liquid-Liquid Partition . . . . . . . 62 Silica Gel Column Chromatography. . . 63 Thin Layer Chromatography. . . . . . . . 65 Densitometric Analysis of Aflatoxins . . 70 Analysis of Aflatoxins in the Feed . . . 72 Analysis of Aflatoxins from Drip of Cooked Meat . . . . . . . . . . . . . 73 Confirmatory Tests for Aflatoxins. . . . 74 Aflatoxin B1. . . . . . . . . . . . . 74 Aflatoxin M1. . . . . . . . . . . . . 75 General Confirmatory Test for Aflatoxins . . . . . . . . . . . . . 76 Three-Dimensional Chromatography. . . 76 Preparation of Aflatoxin Reference Standards. . . . . . . . . . . . . . . . 79 Fat and Moisture Analysis . . . . . . . . . 79 Moisture Content . . . . . . . . . . . . 79 Fat Content. . . . . . . . . . . . . . . 79 iv Safety Procedures RESULTS AND DISCUSSION . Feeding Trial Gross Observations on Tissues Aflatoxin Residues in the Tissues of Pigs Fed a Contaminated Diet Withdrawal Trial Effects of Processing and Cooking upon the Levels of Aflatoxins in Pig Tissue Broiling of Fresh Tissue Processing of Hams Processing of Bellies. SUMMARY AND CONCLUSIONS APPENDIX A STATISTICAL ANALYSIS Analysis of Variance APPENDIX B SUPPLEMENTAL TABLES LIST OF REFERENCES Page 80 82 82 88 91 104 113 113 114 114 121 125 127 136 TABLE 10 11 LIST OF TABLES Ratios of Aflatoxin B1 Levels in the Feed in Relation to Aflatoxin B1 or M1 Levels in Edible Tissues. Composition of Ration. Smoking-Cooking Schedule for Bacon andHam. Summary of Feeding Trial for Pigs from Trials 1 and 2 . Average Feed Intake, Average Afla- toxin Consumption, Feed Efficiency and Daily Dosage Ratio for Pigs on Trials 1 and 2 - . - Summary of Organ Weights Expressed as Percent of Live Body Weight for Pigs on Trials 1 and 2 . . Aflatoxin Residues Gag/kg) Detected in Selected Pig Tissues at Zero Time Interval Following Removal of Contaminated Diet. - - - - - . Aflatoxin Residues Gag/kg) Found in Selected Tissues of Control Pigs from Trial 1.. . . . . . Ratios of Aflatoxins B1 or B2 Levels in the Feed in Relation to Aflatoxins M1 or M2 Levels in .Kid- neys and Livers Average Calculated Value for Afla- toxins Found in Selected Pig Tissue . - - Aflatoxin Residues (Ag/kg) Detected in Selected Pig Tissues at One Day Interval Following Removal from the Contaminated Diet. . . . . . vi Page, 36 56 58 84 85 86 92 95 98 101 105 TABLE 12 13 14 15 16 17 18 19 20 21 Aflatoxin Residues (ug/kg) Detected in Selected Pig Tissues at Two Days Interval Following Removal From the Contaminated Diet. . . Aflatoxin Residues Gag/kg) Detected in Selected Pig Tissues at Four Days Interval Following Removal From the Contaminated Diet . Aflatoxin Levels (Ag/kg) Expressed as Dry Weight Basis Found in Pig Tissues Processed in Different Ways. Performance Data for Six Weeks Feedin Experiment of Pigs From Trial I Slaughtered at Zero Day Withdrawal . - Internal Organ Weights in Grams and as Percentages of Body Weight (Values in Parenthesis) of Pigs from Trial 1 Slaughtered. at Zero Day Withdrawal . Performance Data for Six Weeks Feeding Experiment of Pigs from Trial 2 Slaughtered at Zero Day Withdrawal . . Internal Organ Weights in Grams and as Percentages of Body Weight (Values in Parenthesis) of Pigs . from Trial 2 Slaughtered at Zero Day Withdrawal . - - . . Aflatoxin Levels (Ag/kg) Detected in Raw Bellies, Raw-Cooked Bellies, Smoked Bacon and Smoked—Cooked Bacon Expressed as Dry Weight Basis- Aflatoxin Levels (kg/kg) Expressed as Dry Weight Basis Found in Raw Loin Tissues and Raw-Cooked Loin Aflatoxin Levels (Ag/kg) Expressed as Dry Weight Basis Found In Hams Processed in Different Ways. vii Page 107 108 115 127 128 129 130 131 132 133 TABLE 22 23 24 Moisture Determination Data for Raw Loin and Cooked Loin Samples , Moisture Determination Data for Raw Ham, Raw-Cooked Ham, Cured- Smoked Ham and Cured-Smoked-Cooked Ham . . . . . . . . . . . . Moisture and Fat Data for Raw Belly, Fried Belly, Smoked Bacon and Fried-Smoked Bacon . . . viii Page 132 134 135 LIST OF FIGURES FIGURE Page 1 Structures of aflatoxins Bl, B2, G1 and G2 . . . . . . . . . . . . . . . 13 2 Structures of aflatoxins M1 and M2 . . . 15 3 Structural formulas for aflatoxin B1 metabolites . . . . . . . . . . . . . . 20 4 Partial structures for aflatoxin B1 metabolism showing two routes for metabolic activation to the hemiacetal (Bza) and the 2,3-oxide (epoxide). . . . . 25 5 Spotting and Scoring Pattern for Two- Dimension 10x10 cm TLC Plates. . . . . . 66 6 Spotting and Scoring Pattern for Two- Dimension 10x20 cm TLC Plates. . . . . . 67 7 Spotting and Scoring Pattern for Two- Dimension 20x20 cm TLC Plates. . . . . . 68 ix INTRODUCTION The aflatoxins are a group of closely related metabo- lites produced by Aspergilli, principally A. flavus and A. parasiticus. These metabolites can occur as natural contam- inants in animal feeds, as well as in a wide variety of food material used for human consumption. Experimentally, aflatoxins have been shown to be potent hepatocarcinogens, and epidemiologically they are believed to be important human carcinogens. Aflatoxicosis, the dis- ease caused in animals upon consumption of aflatoxin-contam- inated feeds, has been described extensively in the litera- ture in connection with presumptive poisoning of experimental animals, such as cattle, swine, turkeys, ducks and other animals. Effects of aflatoxins in_vivg vary with the dose, the duration of exposure, the species and the nutritional status of the animal affected. The aflatoxins may be acutely toxic when given in large doses, whereas, sub-lethal doses produce chronic toxicity and low levels of chronic exposure may re- sultzhncarcinogenesis. The LD5O values for most species of animals varies from 0.5 to 10 mg/kg body weight. The transmission of aflatoxins through farm animals to the human food chain is very important since studies have shown that ingested aflatoxins may be deposited as the orig- inal aflatoxin, or as one of its metabolites. The levels of aflatoxins found in the tissues are far lower than the levels found in the contaminated feed EEE.§E¢ However, the risk from chronic response of humans from eating contaminated meat can not be under-estimated since prolonged exposure to low levels of aflatoxins in the diet can cause liver tumors in a number of species, including trout, ducklings and rats. Trout and some species of rats, which are very susceptible to the carcinogenic effect of aflatoxins, develop liver tumors upon exposure to only a few parts per billion. Studies on metabolism and tissue deposition of afla- toxins in different animal species have shown that most in- gested aflatoxins are excreted within 24 hours, either as the original aflatoxin or as one of its metabolites. Accord- ing to these studies most aflatoxins are biotransformed by the cytoplasmic or microsomal enzymes in the liver to more polar derivatives, which can undergo further conjugation with endogenous compounds, such as the active forms of glu- curonic acid, sulfate, glutathione and amino acids. The aflatoxin metabolites have increased water solubility and are more efficiently removed from the body than the original aflatoxin. The present study was designed to investigate various means of decreasing the levels of aflatoxins in the tissues of pigs and in meat products prepared from contaminated tissues. The amounts and kinds of aflatoxins carried over into the tissues of pigs fed on an aflatoxin-contaminated diet were determined by analyzing the tissues at zero days withdrawal. The remaining pigs were placed on the uncontam- inated control diet and killed at different time intervals, to determine the length of time required for tissue clear- ance. Finally, contaminated tissues were used to determine the effects of curing, smoking and cooking upon the levels of aflatoxins in hams and bacons. REVIEW OF LITERATURE Occurrence of Aflatoxins The term mycotoxin is derived from the Greek words mykes meaning fungus and toxicum meaning poison or toxin (Goldblatt, 1972). Thus, the term literally means toxins from fungi. All fungi, including Aspergilli produce a large number of metabolites, which according to Steyn (1977) can be divided roughly into two categories: (1) molecules of primary metabolic and structural importance to the organism, and (2) those performing secondary or no obvious functions in the cells producing them. Mycotoxins are included under the latter category. Unlike bacterial toxins, mold toxins are not proteins, and in comparison to botulinal and other bacterial toxins, their chemical structure is simpler (Hesseltine, 1979). As shown by Bu'Lock (1975) and cited by Steyn (1977), the mycotoxins are genotypically specific and are produced by a consecutive series of enzyme-catalyzed reactions from a primary pool of small molecules, such as acetate, malonate, mevalonate, pyruvate and amino acids. According to Weinburg, (1971) the most probable role of the secondary metabolites is for insuring an orderly disposal of intermediates that accumulate when the cell stops dividing. The process provides a safety valve, without which resting cultures of cells would die. Many secondary metabolites are extremely toxic to other forms of life. For example the antibiotics fall under this category. Others, such as the mycotoxins, are muta- genic, carcinogenic or teratogenic (Hesseltine, 1979). There is some question involving the magnitude of the dis- Teases caused by mycotoxins. Recently, Hesseltine (1979) reviewed the occurrence of diseases in man and animals since the beginning of recorded history, and concluded that many diseases were caused by the metabolites of fungi. The aflatoxins are a group of closely related meta- bolites produced by Aspergilli, principally by A. flavus and A. parasiticus (Hesseltine, 1968). They are by far the most extensively studied and the most important of the mycotoxins. Experimentally, aflatoxins have been shown to be potent hepatocarcinogens (Lancaster, 1961; WOgan, 1973). Epidemio- logically, they may represent important human carcinogens (Campbell and Stoloff, 1974). Aflatoxicosis , the disease caused in animals upon consumption of aflatoxin-contaminated feeds, has been described extensively in the literature in connection with presumptive poisoning of experimental ani- mals, such as cattle, swine, turkeys, ducks and a host of other animals (Newberne and Butler, 1969). The discovery of aflatoxins started in England in the early 19603 with a severe toxic outbreak of a condition, which became known as turkey X disease because of the loss of at least 100,000 turkey poults (Blount,1961). In addi- tion, ducklings and other young farm animals were also af- fected (Asplin and Carnaghan, 1961). Generally, the affected birds suffered loss of appetite, showed lethargic signs and wing weaknesses before dying. In most cases, postmortem examination revealed hemorrhages or pale necrotic lesions in the livers, and frequently engorged kidneys. Brazilian groundnut (peanuts) meal in feed given to birds was suspected to be the poisoning agent (Blount, 1961). A similar incident occurred with poultry feed con- taining Brazilian groundnut meal in East Anglia, where 14,000 ducklings died within 4-5 weeks. No fatal cases, however, were observed on a ration from which the groundnut meal was removed (Asplin and Carnaghan, 1961). Later an outbreak of a disease occurred in pigs, which was apparently caused by an unknown toxic factor (Loosmore and Harding, 1961). A similar disease in cattle, reported to be indis- tinguishable from ragwort poisoning, was deScribed the same year by Loosmore and Markson (1961). The cause of disease in these animals was traced to the presence of Brazilian groundnut meal in their rations (Loosmore and Markson, 1961). The possibility that groundnut toxicity was not re- stricted to Brazilian groundnut meal was confirmed by Sargeant 2E.él- (1961b), who gathered samples from.13 other _countries. They detected toxicity in the samples from India, Uganda, Tanganyika, French West Africa, Nigeria, Gambia and Ghana. Toxicity was also observed with maize meal by Allcroft and Carnaghan (1963) and in cotton seed cake by Loosmore EE,E1- (1964). Lancaster gt El- (1961) fed rats with 20 percent Bra- zilian groundnut meal in a purified diet for 6 months and obtained multiple liver tumors in 9 out of 11 animals. Two animals also developed lung metastasis. These tumors did not occur in association with cirrhosis, cell necrosis, or cellular infiltration, thus it was assumed that the toxic agent directly affected the liver cells.l A fatal disease (exudative hepatitis) was observed in guinea pigs by Paget (1954). Symptoms of the disease were manifested by the appearance of small lesions on the liver, pancreas and lymphoid tissues, and production of ascites. However, the cause of the disease was attributed to a nutri- tional deficiency (Paget, 1954). The same disease was reported by Stalker and McLean (1957), and they suggested that it might be due to some contaminant in the diet. These reports were later shown by Patterson SE El- (1962) to be the earliest cases of aflatoxin poisoning. A. flavus had been implicated earlier as a producer of mycotoxins by Kulik (1957), who reported that extracts from peas contaminated with A. flavus were toxic to cats and rab— bits. A. flavus was then implicated in the poultry myco- toxicosis when Forgacs 95 El- (1958) isolated a toxin-pro- ducing strain from grain, which produced the "hemorrhagic syndrome" in poultry. A disease in dogs, referred to as hepatitis X (Seibold, 1953) was investigated by Newberne £5 31- (1955). It was traced to a diet containing peanut meal. Although the dis— ease was reproduced by feeding the toxic meal, the exact nature of the etiological agent was not elucidated. Later, a fatal disease in swine and cattle characterized by liver lesions was reported by Burnside 2E.él- (1957), who associ- ated the disease with the incidence of moldy corn in the feed. They were able to isolate pure cultures of toxic strains of A. flavus and also of Penicillium rubrum. They found that sterile corn spiked with these molds and fed to animal produced similar symptoms to those observed in the disease, but the emphasis at this time was placed only in the P. rubrum.culture. Bailey and Groth (1959) later showed that the moldy corn poisoning of swine was the same as hepatitis X disease found in dogs. Wilson SE 31. (1967) described a similar toxicosis in swine produced by the administration of crys- taline aflatoxins. Isolation of Aflatoxins Sargeant EE.§l' (1961a) extracted the toxic principle in samples of Brazilian ground meal, and concentrated it 250 times. They used exhaustive soxhlet extraction of the sam- ple with methanol, followed by further extraction of the methanol extract with chloroform and then defatted the final extract with petroleum ether. The final extract was fed by intubation to turkey poults and ducklings and produced his- tological liver lesions identical to those seen in field outbreaks of turkey X disease. The identity of the toxic substance in groundnut meal was unknown at this time, but Sargeant 2E.él- (1961a) strong- ly suggested that it was of fungal origin. Furthermore, they ruled out the possibility that the toxic substance was a pyrrolizidine alkaloid or its N-oxide. The toxic princi- ple was further purified by the same group (Sargeant gt 21-» 1961b) using alumina chromatography. This step yielded colorless crystals. Although the preparation was not yet pure, it fatally affected 1-day-old ducklings within 24 hours on administering a dosage of ZO/Ag. Considerably less produced the characteristic histological liver lesions. The crystaline product isolated by Sargeant Ag El- (1961b), when chromatographed on Whatman no. 1 paper with 5 percent acetic acid in n-butanol, gave a single spot with an Rf value of 0.7, and emitted a bright-blue fluorescence under UV light. Of great significance was the fact that the amount of mate- rial present estimated visually corresponded with the toxic- ity of the samples as determined biologically (Sargeant gt §l°» 1961a). Thus, a chemical assay for aflatoxin was developed. Sargeant 35 £1. (1961b) finally confirmed that the toxin was of fungal origin. First, they produced pure cul- tures of certain of the fungal species present in highly contaminated samples of peanuts. When an extract of 10 A. flavus was chromatographed on paper, a fluorescent spot 'with an Rf value of 0.7 was obtained. The material from this spot was toxic to ducklings and produced symptoms associated with turkey X disease. Therefore, Sargeant EE.§l~ (1961b) used paper chromatography to isolate a fluorescent material with an Rf value of 0.7 from the toxic peanut meal. It was then found to be identical to the material present in exe tracts from pure cultures of A. flavus. The toxic material was named aflatoxin in view of its origin. It was soon found that the toxin obtained after paper chromatography was still a complex mixture. Nesbitt 35 El- (1962) using alumina chromatoplates with chloroformemethanol (98.5:l.5) were able to resolve the material from paper chromatography into two fluorescent spots under UV light. One had an Rf value of approximately 0.6 and exhibited a violet-blue fluorescence, while the other migrated slightly more slowly-and exhibited a green fluorescence. For conve: nience, these authors referred to them as aflatoxin B and G, respectively. Nesbitt 2E 31. (1962) also reported the melting points, chemical formulas and molecular weights, along with other physical and chemical characteristics of the aflatoxins B and G by using ultraviolet and mass spec- trometry. De Iongh Ag El- (1962) used silica gel column chroma- tography to purify a crude extract of groundnut meal iso- 1ated by the procedure of Sargeant gE_A1. (1961a,b). The crude extract was sequentially eluted with chloroform and 11 ‘methanol. Aflatoxins were only detected in the chloroform fraction, which was dried and transferred to Kieselgel TLC plates and developed with chloroform: methanol (98:2). This resulted in several spots, which exhibited different fluores- cent colors under UV light. They named the different spots F31, FBZ' FB3 etc. When the extracts of the spots from sev- ' eral plates were administered to ducklings alone or in come bination together, some differences in the degree of toxicity were found. Smith.and McKernan (1962) also used silica gel Kiesel- gel G for the chromatographic separation of aflatoxins frmm extracts of highly toxic strains of A. flavus. They used chloroformemethanol-formic acid (95:5:1) as the solvent and separated at least 12 clearly defined spots, which fluoresced under UV light. Five of the spots caused the characteristic liver lesions in ducklings. These authors also introduced a confirmatory spray test for aflatoxins based on the change in fluorescence of the aflatoxins under UV light. The color changed from a blue or green color to a bright, yellow color when the chromatograms were sprayed with 50 percent sulfuric acid. Hartley EE.§l‘ (1963) were the first to report the isolation and characterization of the four main aflatoxins, which they named aflatoxin 31’ B2, G1, and G2. A crude mix- ture from sterilized groundnut meal, which had been previ- ously inoculated with a toxin producing strain of A. flavus, was resolved into several fluorescent spots. They used 12 silica gel G and chloroformdmethanol (98:2) as the solvent. The four aflatoxins were identified on the chromatoplates, and further isolated and purified using silica gel G column chromatography. They concluded that the materials previously described as aflatoxin G (Nesbitt gt gt., 1962) and aflatoxin FBl (De Iongh gt gt., 1962) were identical to aflatoxin G1 ' and Bl’ respectively. Hartley gt gt. (1963) also demons strated that the material originally called aflatoxin B by Nesbitt gt gt. (1962) was a mixture of aflatoxin B1 contam- inated with aflatoxin B2. Hartley gt gt. (1963) reported the isolation of the, four main aflatoxins, Van der Merwe gt gt. (1963) demonstrated that aflatoxin Bz is the dihydro-derivative of aflatoxin B1. They synthesized aflatoxin B2 by catalytic hydrogenation of aflatoxin B1 with the uptake of one molar equivalent of hydrogen. Vander Merwe gt gt. (1963) also showed that afla- toxin G2 is the dihydro-derivative of aflatoxin G1 and can be produced in the same manner. They then put forward ten- tative structures for aflatoxins B1 and G1. The true struc- tures of the aflatoxins, which differed slightly from those put forward by Van der Merwe gt gt. (1963), were finally elucidated and confirmed later by further studies (Asao 2E.il°» 1963, 1965). The structures of aflatoxins B1, Bz, G1 and G2 are shown in Fig. l. The first indication of occurrence of aflatoxins other than B1, B2, G1 and G2 Was reported by Allcroft and Carnaghan (1963). They found that extracts of milk from cows fed 13 o o \ loJ‘o / OCH; AF. 3. o o LoJ‘o I / OCH3 AEBZ AFGZ Figure 1 - Structures of aflatoxins B1, 32, G1 and G2. l4 aflatoxin containing toxic groundnut meal induced liver lesions identical to those caused by aflatoxin B1. TLC exam- ination showed that there was no aflatoxin B1 present. The milk toxin, as it was named, was shown to be identical to a bluedviolet fluorescent component also present in toxic groundnut meal (De Iongh gt gt., 1964). Allcroft and Carnaghan (1963) concluded that the toxic factor in milk resulted from metabolism of aflatoxin Bl by the animal rather than from direct ingestion, since rats fed pure afla- toxin Bl excreted the same metabolite found in milk. Allcroft gt gt. (1966), also isolated the milk toxin from the urine of sheep fed aflatoxin B1 and confirmed its chromatographic equivalence to the milk toxin. They further proposed the generic name aflatoxin M for the toxin found in the milk. Holzapfel gt gt. (1966), repeated the experiments of Allcroft gt gt. (1966), isolated aflatoxin M from the urine of aflatoxin-dosed sheep and separated it into two Components using paper chromatography. The blue-violet component was named aflatoxin M1 and the violet spot aflatoxin M2. On the basis of ultraviolet, infrared, nuclear resonance and mass spectral data, these authors identified the structures of aflatoxins M1 and M2. Masri gt gt. (1967) later confirmed the structure of M1‘ It was postulated that aflatoxin M1 was the hydroxylated derivative of aflatoxin B1, with the hydroxyl group in the C-4 position of the terminal furan ring, and that aflatoxin M2 was the dihydro-derivative of aflatoxin M1 resulting from the hydrogenation of aflatoxin 15 M1. The structures of aflatoxins M1 and M2 are shown in Fig. 2. Figure 2 - Structures of aflatoxins M1 and M2. Two additional forms of aflatoxin were isolated by Dutton and Heathcote (1966) from cultures of A. flavus. They concluded that the two new aflatoxins were hydroxy deri- vatives of aflatoxins B2 and G2, and were thus, named afla- toxin Bza and 32a» respectively. Later they elucidated the structures and biochemical properties. They found that afla- toxins 323 and G2a were much less toxic to ducklings than the other aflatoxins (Dutton gt gt., 1968). Subsequently. improvement of the analytical techniques for extraction of aflatoxins from animal tissue and develop- ment of TLC procedures with suitable solvent systems and the use of HPLC and mass spectrometry have enabled researchers to identify many other forms of aflatoxin metabolites: (1) Afla- toxin Pl - a demethylation product of aflatoxin B1 identified as a major metabolite of aflatoxin B1 in rhesus monkeys l6 (Dalezios EE.§l-» 1971); (2) Aflatoxin Q1 - isolated from post-mitochondrial liver preparations from rat, monkey and humans as well as from monkey urine (Masri gt gt., 1974; Adenkule, 1977; Buchi gt_gt., 1974); (3) Aflatoxicol-iso- lated from postamitochondrial liver preparation of humans, rabbits and several avian species, in addition to being the major metabolite in the plasma of rats dosed with aflatoxin B1 (Patterson, 1973; Patterson and Roberts 1971, 1972a, 1972b; Salhab and Edwards, 1977; WOng and Hsieh, 1978); and (4) Aflatoxin Bl-epoxide-formed through the metabolic epoxidation of the 2,3 vinyl ether double bond of aflatoxin B1. This metabolite has not yet been isolated, probably because of its instability and great reactivity, but studies have strongly supported its formation tg yttg and iE.ZiE£9 by incubation of aflatoxin B1 with post-mitochondrial liver fractions (Garner gt gt. 1971, 1972; Swenson gt gt., 1973, 1975, 1977; Roebuck gt gt., 1978). Metabolism of Aflatoxins Drugs and other foreign compounds enter the body mostly by absorption from the gastrointestinal tract, from which they are taken via the portal vein to the liver, where they may be chemically modified through different types of reactions, such as oxidation, reduction, hydrolysis and con- jugation (Kappas and Alvares, 1975). Their essential effect is to convert lipophilic or fat-soluble compounds into hydro- philic or water soluble substances (Kappas and Alvares, 1975). 17 The modified products may either flow into the bile to be excreted in the feces or to the systemic circulation before being taken to the kidneys and voided in the urine. Other sites of drug metabolism are located in the lungs, kidneys, the skin and the gastrointestinal tract itself (Blumberg, 1978). The microsomal mixed function oxygenase, an enzyme complex located in the endoplasmic reticulum of the cell, catalyzes the metabolism of various drugs, carcinogens, steroids, insecticides, and other compounds (Conney, 1967), including the aflatoxins (Garner gt_gt., 1971, 1972). More than 200 compounds can influence the activity of this come plex and these have been divided in two general classes (Conney, 1967): (1) the phenobarbital type, which enhances the formation of cytochrome P-450 and the activity of mixed function oxygenase toward several substrates; and (2) the polycyclic aromatic hydrocarbon type, exemplified by 3- methylcholanthrene and benzo(a)pyrene, which enhance the activity toward a limited number of substrates and stimulate the formation of different types cytochromes, cytochrome P1-450 (Sladek and Mannering, 1966), and P-448 (Alvares gt gt., 1967). Evidence has accumulated to suggest that liver microsomal cytochrome P-450 is a mixture of several forms that can be identified on the basis of their electrophoretic patterns (Walton and Aust, 1974; Haugen gt gt., 1976), by their specificity for being induced by various chemicals (Conney gt gt, 1973) and by their catalytical and physical 18 properties as partially purified fractions (Ryan gt_gt., 1975; Haugen gt gt., 1975). Aflatoxin B1 is metabolized by the hepatic microsomal mixed function oxygenase system to a group of derivatives, such as aflatoxins M1, Q1, P1, B2a and aflatoxin Bl-epoxide (Campbell and Hayes, 1976). Aflatoxin B1 can also be reduced by a cytoplasmic reductase to afla- toxicol (Wong and Hsieh, 1978). Aflatoxin M1 results from.the ring hydroxylation of aflatoxin B1 at the C-4 position (Fig. 3-pathway 1). It was one of the first aflatoxin metabolites to be discovered. It was first identified in milk from cows fed aflatoxin contam- inated rations, thus aflatoxin M ‘was originally called "milk 1 aflatoxin" (Allcroft and Carnaghan, 1963). Soon after Allcroft and Carnaghan (1963) discovered aflatoxin M1 in milk, its presence was confirmed by De Iongh gt gt. (1964). It was later also shown to be present in the urine of animals (Holzapfel EE.§l:: 1966) and humans (Campbell gt gt., 1970), and in tissues of animals (Allcroft gt gt., 1966) ingesting rations containing aflatoxin B1. Aflatoxin P1 is the phenolic metabolite resulting from the O - demethylation of aflatoxin B1 (Fig. 3-pathway 2). Wogan gt gt, (1967) were the first to suspect that O - de- methylation probably occurred during aflatoxin B metabolism, 1 in that approximately 25 percent of the radioactivity admin- istered as 14C-methoxy-labeled aflatoxin B1 to rats appeared in the respired CO2 within 24 hr. Shank and Wogan (1965) however, using 14C-labeled ring carbons failed to demonstrate 19 its presence in the expired C02, which means that the ring cleavage either does not take place or that the products formed by the cleavage are not fully oxidized. The presence of aflatoxin P1 was later confirmed by Dalezios gt gt. (1971) who identified it as the major metabolite in rhesus monkeys given a single intraperitoneal injection of ring labeled aflatoxin B1. Aflatoxin Q1 is another hydroxylated derivative of aflatoxin B1 and an isomer of aflatoxin Ml, with the hydroxyl on the beta carbon atom of the carbonyl of the cyclopentanone ring (Fig. 3-pathway 3). Aflatoxin 01 has been recently discovered in monkey (Masri gt gt., 1974) and rat (Adekunle gt gt., 1977) by liver incubations of aflatoxin B1, and represents the major tt ztttg conversion of aflatoxin B1 by human liver microsomes (Buchi gt gt., 1974). Aflatoxin 32a or aflatoxin B1 hemiacetal results from the hydration of the 2,3 vinyl ether double bond in the aflatoxin B1 molecule (Fig. 3-pathway 4). This transforma- tion is accomplished readily by liver microsomes of a variety of domestic and laboratory animals (Patterson, 1973). Acid catalyzed addition of water to the vinyl double bond of aflatoxin Bl also leads to the formation of aflatoxin B2a (Pohland gt gt., 1968; Pons gt gt., 1972). Aflatoxicol or aflatoxin Ro results from reduction of carbonyl group in the cyclopentanone ring of aflatoxin B1 (Fig. 3-pathway 5). This pathway is especially prominent in the livers of rabbits and several avian species, however, unlike the previous metabolites, the reduction is not 20 Aflatoxin Epoxide Aflatoxicol (R0) Figure 3.--Structural formulas for aflatoxin B metabolites. Numbers indicate the pathways referled to in the thesis. 21 catalyzed by the microsomal mixed function oxidases, but by a NADP-linked dehydrogenase of the cytosol, which also has l7-ketosteroid dehydrogenase activity (Patterson, 1973; Pat- terson and Roberts 1971; 1972a; 1972b). Evidence for this pathway in post-mitochondrial liver preparations from humans has been recently presented by Salhab and Edwards (1977). Aflatoxicol has also been shown to be the major aflatoxin metabolite in the plasma of rats that are dosed orally or intravenously with 14C-aflatoxin Bl (Wong and Hsieh, 1978). Aflatoxin B1-2,3-oxide results from the metabolic epoxidation of the 2,3 vinyl ether double bond of aflatoxin Bl (Fig. 3-pathway 6). Schoental (1970) was the first to suggest the possibility of formation of aflatoxin B1-2,3 epoxide by hepatic microsomes. They developed this concept by analogy with the metabolic activation of polycyclic aromatic hydrocarbons. Although this labile molecular spe- cies has not been isolated, much indirect evidence has accu- mulated to substantiate its transient existence (Swenson gt gt., 1973; Croy 95 gt., 1978). Toxicity of Aflatoxins A central issue in the toxicology of aflatoxins is the question of whether their biological activities are due to the effects of the toxins themselves, or indirect,.and a consequence of structural alterations of the toxins pgt gg during metabolism. According to Patterson (1973), experi- mental animals vary considerable in their abilities to 22 metabolize aflatoxins; the diversity of response suggesting that variation in metabolism may be important in determining the toxic action of aflatoxin B1 in different species of animals. Wong and Hsieh (1976) have studied the relative muta- genic potency of aflatoxins using the Ames test (Ames gt a1., 1975). They found that aflatoxin M1 has only about 3 percent of the mutagenic potency of aflatoxin B1. Except for afla- toxicol, which had 23 percent of the potency of aflatoxin 81’ all other metabolites were weaker mutagens than aflatoxin M1. In the same study, Wong and Hsieh (1976) also reported that the activity of the aflatoxin metabolites, as found by the tg_ztttg mutagenicity assay, corrEIated with their tg ttzg carcinogenic activity. Recent studies on the metabolism and mode of action have provided evidence that aflatoxins require metabolic activation to elicit their carcinogenic effects. Wong and Hsieh (1976), using a Salmonella typhimurium mutant assay (Ames gt gt., 1975), have demonstrated that neither aflatox- icol nor aflatoxins M1, Q1, or B a possess activity in the 2 absence of the activation factor from rat liver preparations. This indicated that none of these metabolites are the ulti- mate mutagenic and/or carcinogenic compounds. Magee (1974) reviewed the mechanism of activation of the carcinogenic compounds. He reported that many of these compounds require a metabolic activator to transform an inactive form into its ultimate mutagenic or carcinogenic species. 23 Pathway 6 in Fig. 3 (also shown in Fig. 4) seems par- ticularly important in elucidating the mechanism of metabolic activation of aflatoxin B1. Schoental (1970) first postu- lated that the formation of an epoxide intermediate of afla- toxin Bl at its 2,3 double bond might account for its toxic— ity. Lijinsky gt gt. (1970) presented data showing the formation of nucleic acid and protein bound radioactive come ponents after the administration of 3H-aflatoxin B1 to rats. They suggested that the aflatoxin B1 or its metabolites might be covalently bound to these macromolecules. Garner gt gt. (1971; 1972) demonstrated that incubation of rat hepatic microsomes with aflatoxin and a NADPH - gen- erating system resulted in the formation of a reactive metab- olite that was toxic to certain bacteria. The metabolite was not isolated but the inclusion of nucleophiles in the incubation medium, such as DNA and RNA, increased the sur- vival of bacteria. Inhibition of the toxicity of the afla— toxin B1 metabolite by the nucleic acids in the liver micro- some mediated system suggested that RNA.and DNA might cova- lently bind to the metabolite, since the complex did not dissociate when isolated and passed through a Sephadex col- umn that would free any adsorbed aflatoxin. Garner and Wright (1973) observed an increase in the amount of the lethal aflatoxin B1 metabolite formed by phenobarbitone induced microsomes over those induced by 3- methyl cholanthrene or benzo(a)pyrene using the bacterial survival assay (Garner gt gt., 1971, 1972). Gurtoo and 24 Bejba (1974), using a different approach, reported that the epoxidation of aflatoxin B1, measured as a DNA-alkylating metabolite, is enhanced by pretreatment of rats with pheno- barbital, but not by Bdmethyl cholanthrene. They also found that the addition of cyclohexene oxide, an epoxide hydrase inhibitor, did not increase binding of aflatoxin B1 to form DNA adducts, but the possibility of an epoxide intermediate was not ruled out. Similarly, Garner and Wright (1973) did not detect any alteration in bacterial inhibition when cyclohexene oxide was added to the assay. More evidence for the formation of the reactive afla- toxin Bl derivative was reported by Swenson EE.§1- (1973). They showed that on incubating aflatoxin B1 tt ytttg with rat and hamster liver microsomes and RNA, a nucleic acid adduct was formed. Mild acid hydrolysis of the nucleic acid adduct liberated derivatives that were indistinguishable from 2,3-dihydro-2,3-dihydroxy-af1atoxin B1 (Fig. 4). These observations strongly support the concept that aflatoxin Bl .is metabolically converted to an aflatoxin Bl-2,3-oxide, which binds covalently through its high electrophylic C-2 to nucleophilic sites on nucleic acids, probably at the N-7 or 0 position on the nucleotide residue. This hypothesis has been recently confirmed by Croy gt gt. (1978), who iso- lated the 2,3-dihydro-2-(7N-guanyl)-3-hydroxy-af1atoxin B1 as the principal product after hydrolysis of liver DNA of rats dosed with aflatoxin B1. These findings are in perfect agreement with Miller 25 .Annaa .aomumuummv vmumowoaw omam mum mcownu Imusaw poosvou nuaa sowuomon ofiuma%uao an ovwxoao onu wo soaum>fiuomsfi was 92¢ cam Gaououm sues moufiaoomuoa omega mo maofiuomuousH .Aopfixoeov opwxo m.N mnu cam Ammmv Hmuoomfiaon onu ou coflum>wuom owaonmuoa How mousou osu weasonm amHHonmuoE Hm saxoumamm How mousuosuum Hmfluumm I q ouswam Hosaa< <29 /_\oJ\o/_Iezo aopnm< MW _ o: monme-0.05 ppb) in 63 percent of all southeastern U.S. samples, in 80 percent of the samples from the state of Georgia, and in one or more samples from 84 percent of all bottling plants in the sample area (Stoloff, 1980). In Arizona in 1978, milk from cows fed cottonseed meal containing aflatoxins at the ppm level was found to have apreciable amounts of aflatoxin M1 (Stoloff, 1980). Similar incidents were observed in 6 adjacents states due to the use of contaminated cottonseed meal before State and Federal regulatory authorities could control the situation (Stoloff, 1980). This culminated in the FDA establishing a maximum allowance of 0.5 ppb for aflatoxin M1 in fluid milk (FDA 1977). Stability of Aflatoxins in Foods Although aflatoxins are fairly stable molecules, heat processing and storage may reduce the initial levels found in foods. Coomes gt gt. (1966) reported that autoclaving moist peanut meal for 4 hours at 120° C reduced the amount of aflatoxin from 7,000 to 350 ppb. .Biological assay of the autoclaved meal showed a corresponding decrease in tox- icity. In the same study, Coomes gt gt. (1966) autoclaved pure aflatoxin B1 for 4 hours at 120° C. They also refluxed pure aflatoxin B1 with water for 10 hours. They then com- pared the isolated products from the two model systems with the material formed after roasting. Based on the UV spectra 49 of the isolated compounds, they conclude that heating afla- toxin Bl in presence of moisture causes hydrolytic opening of the lactone ring on the aflatoxin B1 molecule, which can further be decarboxylated, leading to non-toxic breakdown products. Mann gt gt. (1967) made a more detailed study of the effect of heat and moisture on aflatoxins in oil seed meals. They found that temperatures of 60 to 80° C have little effect on aflatoxins in oil seed meals, while substantial amounts of aflatoxin are degraded at 100° C. They reported that increasing the moisture content and/or the heating time caused a proportional decrease of aflatoxins in the meal. For example, heating meal containing 20 percent moisture for 2 hours at 100° C resulted in degradation of approximately 80 percent of the aflatoxin. Reducing the time to 1 hour at 100° C resulted in doubling of the residual aflatoxin level. Reducing the temperature to 80° C and heating for 1 hour re- sulted in a further doubling of the residual aflatoxin level. Peers and Linsel (1975), however, observed that aflatoxin B1 was extremely stable in peanut and corn 011. They found that aflatoxin B1 was not degraded in these oils until dratamxnammm: approached 250° C, which is close to the melting point of aflatoxin B1. Lee gt gt. (1969) studied the stability of aflatoxins under conditions simulating commercial dry and oil roasting of peanuts artificially contaminated with 130 to 6300 ppb of total aflatoxins. They found an average reduction of afla- toxin concentration ranging from.45 to 83 percent, depending on roasting conditions and initial aflatoxin levels. Oil 50 roasting times varied from 3 to 7 minutes with the tempera- tures varying from 325 to 345° F. Dry roasting times varied from 5 to 30 minutes, with the temperatures varying from 250 to 400° F. Similar results were obtained by Waltking (1971) upon roasting commercially rejected peanuts on a pilot plant scale under conditions simulating those used for making pea- nut butter. The aflatoxin levels for raw peanuts varied from 500 to 627 ppb of total aflatoxins. They reported an average loss of 40 to 50 percent and 20 to 40 percent for aflatoxins B1 and G1, and B2 and G2, respectively. Conway gt gt. (1978) also obtained similar results roasting aflatoxinecontaminated corn. They showed a 40 to 80 percent reduction in aflatoxins could be obtained by a single passage of corn through a continuous roaster (temper- atures ranging from 145 to 165° C). In a second experiment they combined the effects of the heat and ammonia treatments. They reported a reduction of 57 percent in the aflatoxins from corn tempered to 20 percent moisture with an aqua ammo- nia concentration of 0.5 percent NH3 on a dry weight basis when passed through the corn roaster. When the corn was re- tempered as described above and again passed through the roaster, a further reduction in the aflatoxin concentration resulted. Redu'ctiOns of over 90 percent were achieved by this process. Ammoniation has been sucessfully used in the decontam- ination of several agricultural products contaminated with aflatoxins. A full account of the use of ammonia and other 51 methods of detoxification of aflatoxin-contaminated products are given by Marth and Doyle (1979). Allcroft and Carnaghan (1963) reported that toxic milk from cows fed rations containing 20 percent toxic peanut meal did not show a reduction in toxicity after treatment by pasteurization or by roller-drying. They used the one-day- old duckling assay test (Asplin and Carnaghan, 1961) to measure the toxicity of the milk before and after processing. However, the duckling assay test for aflatoxin M1 is not a particularly sensitive test (Wogan, 1964). Similar results were obtained by Stoloff gt gt. (1975) and Van Egmond gt gt. (1977), who observed no loss of aflatoxin M1 from milk after different forms of heat treatments. In contrast to these reports, Purchase gt gt. (1972) showed that processing of milk reduces its aflatoxin M1 content, and that the higher the temperatures used the lower the amount of aflatoxins. Using chemical analyses, they reported a reduction of afla- toxin M1 in milk of 33 percent by pasteurizing at 62° C for 30 minutes, and 80 percent upon sterilization at 80° C for 45 seconds. Similarly, they reported a reduction in the levels of aflatoxin M1 from freeze-dried and spray-dried milk, as indicated by both chemical and the ducklings assay tests. McKinney gt gt. (1973) reported that aflatoxin M1 in liquid raw milk disappears very rapidily during storage. They found that in samples with low aflatoxin M1 levels, approximately 40 percent of the initial aflatoxin M1 was not 52 detectable after a period of 4 days, and approximately 80 percent disappeared during 6 days storage at 0° C. However, Stoloff gt gt. (1975) did not observe any decrease of afla- toxin Ml levels in raw milk stored over a period of 17 days at 4° C. On the other hand, they observed a 45 percent reduction of aflatoxin M1 in milk after a period of 120 days frozen storage (-18° C), with detectable changes starting at 68 days. McKinney gt gt. (1973) found an 87 percent reduc- tion of aflatoxin M1 in milk after 120 days storage under similar conditions, with detectable changes starting at 30 days. Strzelecki (1973) reported that recovery of aflatoxin from raw ham, cured ham and salami decreased with storage time. Using different time periods and storage conditions, they found recoveries of 19 percent from salami, 16 percent in raw ham, and 7 percent for cured ham with the amount retained being related to the aflatoxin level added initially to the products. Murthy gt gt. (1975) also reported that there was a decrease in recovery of aflatoxin B1 injected into beef with increased holding periods during storage. The total recovery of injected aflatoxin B1 dropped from.98 percent after 20 days of storage to 79 percent after 183 days. They suggested that incomplete extraction due to inter- actions of aflatoxins with the meat constituents during storage probably accounted for the losses. EXPERIMENTAL Feeding Trial Preparation of the Diet In order to prepare the spiked ration, the standard aflatoxins were first extracted with chloroform and were then diluted to 2 liters in a volumetric flask. The chloro- form solution was then divided into two equal fractions and each of them.was slurried with 1 kg of finely ground feed (screened through a 20 mesh screen and dried over night in an oven at 100°C). The chloroform was allowed to evaporate in the dark over night with forced air under a hood. Each sample of aflatoxin spiked feed was then homoge- nously mixed with additional feed in a 4-speed Reynolds Mixer (Reynolds Electric Co.) to give a final weight of about 7 kg of feed. Then each of the aflatoxin premixed feed samples was separately transferred to a stainless steel wenger hori- zontal mixer (Wenger Mixer Mnfg.Co.), and homogenously mixed with additional feed to give a final weight of about 49 kg. The 49 kg lots were then transferred to a horizontal mixer (Bryant Poff. Inc.) and mixed with additional feed plus the vitamin and mineral supplements to give a final weight of 1400 kg. The two lots of aflatoxin-spiked feed totaled 2800 kg. The feed was packed in 23 kg bags and stored at 53 54 approximately 0° C until fed. The basal ration used for the control group was handled the same, except that it was not spiked with aflatoxins. Experimental Animals Forty crossbred pigs were used in two trials to denamfine the amount of time necessary for tissue clearance after feeding an aflatoxin contaminated diet. The pigs were housed in pens with aluminum slotted floors in an atmOSphere con- trolled building. Each pen was equipped with a self-feeder and nipple-type waterer. Feed and water were offered gt libitum. The pigs were weighed at 7 day intervals except for the last weigh period, which was 6 days in length. Feed consumption was also recorded weekly. The basal diet consisted of corn and soybean meal and was fortified with vitamins and minerals. The composition of the diet is given in Table 2. There were 20 pigs in each trial, with 4 being fed the control diet and 16 being used to determine the time neces- sary to obtain tissue clearance after removal from the con- taminated diet. The spiked diets contained 551 and 355;:g of aflatoxin B1 and Bz per kg of feed, respectively. Since we had not previously found aflatoxins on analysis of the feed, the basal ration was assumed not to contain any con- tamination. However, analysis revealed that the basal diet on trial 1 contained 20 and 31,ug of aflatoxin B1 and 32 per kg of feed, respectively. In trial 2, the basal diet was uncontaminated with aflatoxins. The analysis of feed for 55 aflatoxins was carried out using a modification of the A.O.- A.C. method (1975). In trial 1, preliminary data on the length of time required to obtain tissue clearance after removal of the aflatoxin contaminated diet was determined. The pigs aver- aged 9.6 kg initial weight and they were randomly assigned in three groups based on litter, weight and sex. There were 4 pigs in the control group and 8 pigs in each of the two experimental groups. At the end of the experimental feeding period four experimental pigs and four controls were slaugh- tered. The remaining twelve pigs were placed on the uncon- taminated control diet and held for 1,2,4,8,l6 and 32 days. At the end of each period two pigs were killed and the tis- sues were examined for aflatoxins. Once the length of time for tissue clearance was estab- lished a second trial was carried out using larger members of animals to confirm.the previous results and to give more detailed information. The pigs on trial 2 averaged 9.6 kg initially and they were assigned into four groups according to litter, weight and sex. There were 4 pigs on the control group with 5,5 and 6 pigs in the experimental groups. As in trial 1, four controls and four experimental animals from trial 2 were killed after 42 days of feeding an aflatoxin-contaminated diet. The re- maining 12 pigs were fed an aflatoxin-free diet for 1,2 and 4 days. After each period, 4 pigs were killed and the tis- sues were examined for aflatoxins. Analysis of the tissues 56 for aflatoxins were carried out using a modification of the method of Trucksess and Stoloff (1979). Table 2 - Composition of the Rationa’b Ingredients Percentage CORN-GROUND 75.35 SOYBEAN MEAL 21.85 MINERAL MIXTUREC 2.30 VITAMIN PREMIXd 0.50 aFeed analysis: protein, 16.5%; lysine, 0.80%; methionine + cystine, 0.55%; trypt0phan, 0.19%; calcium, 0.67%, and phosphorus, 0.505%. ‘bDigestible energy, 3436 kcal/kg. CComposition of mineral mixture as percentage of diet: Sod- ium chloride, 0.50; limestone, 1.00; dicalcium phosphate, 1.00; and the following in ppm: Se, 0.1; Zn, 74.8; Mn, 37.4; I, 2.7; Cu, 9.9; and Fe, 59.4. dThe vitamin premix supplied the following per kilogram of ration: vitamin A, 3300 IU; vitamin D, 660 IU; vitamin E, 5.5 IU; vitamin K compound, 2.2 mg; riboflavin, 3.3 mg; niacin, 17.6 mg; D-pantothenic acid, 13.2 mg; choline, 110.0 mg; and vitamin 312, 19.8 Ag. Slaughtering and Collection of Samples The pigs were taken to the MSU Meat Laboratory at approximately 5 p.m. on the day preceeding slaughter. They were held off feed until approximately 6:30 a.m. the fol- lowing day, when they were slaughtered. After slaughtering, the tissues were examined for possible gross lesions by a Michigan State Department of Agriculture Meat Inspector. Samples of blood, heart, kidneys, liver, muscle and spleen were collected from the pigs. The samples were weighed, 57 frozen and stored at -20°C for later analysis. Processing_of Meat Tissues Preparation for Processing Tissue samples were also taken to study the influence of different forms of cooking upon the stability of aflatoxins and to determine the effects of curing, smoking and cooking upon the levels of aflatoxins in hams and bacon. Hams, loins and bellies from.both sides of the carcass from.the 4 exper- imental animals in trial 1 (slaughtered at zero day withdraw- al period) were collected and processed as follows: One of the cuts was divided in two halves, one half was used as the control and the other half was cooked. The other cut was cured, smoked and then divided in two portions, one of which was analyzed before cooking and the other after cooking. Using this procedure the effects of cooking and processing on the level of aflatoxins in the samples were determined by analyzing the meat, both before and after processing. Curing Procedure Ham Curing The fresh hams were stitch pumped to 10 percent by weight. The pickling brine was prepared using the following: 6 lbs salt, 3 lbs sugar, 28 g sodium nitrite and 33 lbs of cold water. The solution was thoroughly mixed before pump— ing into the meat. After pumping, the hams were submerged 58 and held in an identical brine solution for seven days in a cold room at a temperature of approximately 6°C. Bacon Curing The fresh bellies were rubbed with a dry curing mix- ture and placed in plastic boxes inside a cooler at a temper- ature of approximately 6°C. The dry curing mixture was pre- pared using the following ingredients: 6 lbs. salt, 2.3 lbs. sugar and 7 g of sodium.nitrite, per 100 lbs. of meat. The curing ingredients were thoroughly mixed by hand before rub- bing the meat. The bellies were held in the curing room for a period of 7 days to allow for good distribution of the cure throughout the tissues. Smokinngooking.Schedule After the seven days cure, both the hams and bellies were transferred to a Elek-Trol laboratory smokehouse (Dry- ing Systems Inc.). They were then smoked-cooked toéuiinter- nal temperature of 66°C using the schedule shown in Table 3. Table 3 - Smoking-Cooking Schedule for Bacon and Ham Time Temperature (°C) Relative (min.) Dry Bulb Wet Bulb Humidity (%) 135 49 32 30 75 54 38 35 60 60 44 46 165 71 66 76 59 Smoke was applied throughout cooking using a midget size Mepaco smoke generator (Meat Packers Equip. Co.) utili- zing mixed hard wood sawdust. Cooking of Raw Hams In preparing samples for cooking, the raw frozen hams were cut into slices about 2.5 cm thick using a hand meat saw. The frozen slices were thawed and then cooked in stain- less steel pans at an oven temperature of 176°C. The final internal temperature was 76°C. The temperature was monitored by placing a thermometer in the meat tissues. To assure uni- form cooking, the position of the meat pieces inside the oven was switched at regular intervals. The cooked tissues were then ground and stored as described earlier herein. The drip from each sample was collected and stored for subse- quent analysis. Cooking of Cured Ham Samples Slices of cured ham.were prepared similar to those described above. The ham slices were cooked in an electric oven at about 163°C, until an internal temperature of 71°C was reached. The cooked tissues were ground and stored as described earlier. The drip from the various samples was pooled and stored as outlined above. 6O Frying Belly and Bacon Samples The belly and bacon samples were sliced into thin strips about 2.5 mm thick before frying. To assure good slicing, the samples were semi-frozen and then immediately sliced using an electric bacon slicing machine (Hobart Mnfg. Co.). The meat strips were then cooked for 3 minutes on each side using an electric fry pan (Sunbeam Appliance Co.) with the temperature set at 171°C. After cooking, the samples were ground and stored as described earlier. The drip was collected and the frying pan was washed thoroughly after cooking each sample in order to prevent any cross contamina- tion. Broiling of Loin Samples The loin samples were placed in stainless steel pans and cooked with the oven set for broiling. They were then broiled until the t0p of each chop was lightly brown, at which time they were turned over until the other side was browned to the same extent. The internal temperature of the meat was monitored with a thermometer and the samples were removed from the oven at an internal temperature of 76°C. The cooked samples were ground and then stored as described earlier. 61 General Methods of Analysis for Aflatoxins Sample Preparation for Extraction The tissue samples were deboned and the collagenous tissues and excess fat were removed from the lean portion. All tissues, except for the internal organs weighing less than 100 g, were passed twice through a meat grinder (The Hobart Mnfg. 00.), using a plate having 3/16 inches diameter holes. The ground tissues were then thoroughly mixed before removing samples for analysis. The tissues were stored at -20°C until analyzed. The internal organs (kidneys, hearts and spleens) were cut into small pieces without thawing and placed directly in the blender. Extraction of Aflatoxins from Tissues Extraction and analysis of aflatoxins from raw tissues and the processed samples were carried out according tozanodi- fication of the procedure of Trucksess and Stoloff (1979). About 100 g of raw tissue or 60 g of cooked tissue were blend- ed for 2 minutes in a Waring blender at moderate speed with 42 ml of NaCl - citric acid solution (35 g NaCl + 4.8 g citric acid/100 ml H20). Then 300 ml of acetone was added to the homogenate while washing the sides of the blender jar. The tissue was blended for an additional 3.0 minutes at mod- erate speed followed by 2 minutes at high speed. The mate- rialwas then filtered through fast filtering prefolded filter paper (Whatman 114 v), and the filtrate was collected 62 in a 250 ml graduate cylinder. After filtration was com- pleted, the meat residue was discarded. A total of 235 m1 of the filtrate was transferred to a 500 ml Erlenmeyer flask. Then 20 m1 of Pb(OAc)2 solution (200 g Pb(OAc)2.3H2O in 500 m1 H2O containing 3 ml of ace- tic acid and diluted to 1 liter with H20) and 150 ml of H20 were added. The solution was stirred for 0.5 minutes using a magnetic stirring device, and then 10 g of (NH4)ZSO4 were added. The stirring was continued for an additional minute while 10 g of diatomaceous earth was added to the so- lution. The solution was allowed to stand for about 5 min- utes before filtering through fast filtering folded filter paper into a 500 ml graduate cylinder. The residue on the filter paper was discarded. Purification of the Aflatoxin Extract Liquid-Liquid Partition Exactly 325 m1 of the filtrate were transferred to a 500 ml separatory funnel. Then 100 m1 of petroleum ether (BO-60°C b.p.) were added. -The separatory funnel was shaken vigorously for about 1 minute. The layers were allowed to separate, and the lower aqueous-acetone layer was drained into a second 500 m1 separatory funnel. The petroleum ether layer was then discarded. Then 50 ml of chloroform were added to the aqueous-acetone solution and the separatory funnel was shaken as before. After the layers separated, the 63 lower chloroform acetone layer was collected in a 500 m1 flask. Aflatoxin extraction from the aqueous-acetone layer was repeated one more time using 50 ml of chloroform-acetone (1:1). The aqueous layer remaining after the chloroform extraction was discarded. In some of the samples, especially in cooked tissues, a small amount of water was trapped with- in the chloroform-acetone extract. The water was removed by passing the chloroform-acetone extract through a column packed with 10 g of anhydrous sodium sulfate. After the second chloroform—acetone extract was passed through the column as before, it was washed with an additional 50 ml of chloroform. The eluates were collected in a 500 m1 flask. The chloroform-acetone extract was then evaporated to dryness in a rotary evaporator (Buchi, Switzerland) using a water bath setting of 40°C. Silica Gel Column Chromatography Chloroform.was added to a 22x300 mm chromatographic column until the tube was 2/3 full. Then a ball of glass wool was placed in the bottom of the tube and approximately 5 g of anhydrous sodium sulfate were added to give a base for the silica gel column. Then 10 g of silica gel 60 (70- 230 mesh ASTM-EM Laboratories Inc.), which had been previ- ously slurried in 50 ml of chloroform, were added to the col- umn. The silica gel was allowed to settle and then the chlo- roform was drained to about 2 cm above the top of the silica gel. About 2 cm of anhydrous sodium sulfate was layered 64 slowly on top of the silica gel. The excess of chloroform was then drained from the column until it reached the level of the upper layer of sodium sulfate. The aflatoxin extract was dissolved in approximately 5 ml of chloroform-hexane (1:1) and then transferred to the column with a disposable glass pipet. The sides of the flask were washed three more times with 5 ml of chloroform-acetone (1:1) and the washings were added to the column. After each addition of chloroformrhexane extract, the column was drained to the top of the packing and the eluate was discarded. Any interferring substances were eluted from.the column in 100 ml of the ether-hexane (3:1). The eluate was then discarded. The aflatoxins were eluted from the silica gel column with 160 m1 of chloroformrmethanol (97:3). The eluate was collected in a 250 ml flask and evaporated to near dryness in a rotary evaporator as described earlier herein. The sample extract was dissolved in dichloromethane-acetone (1:1) and quantitatively transferred to 2 dram vials using a dis- posable glass pipet. The dichloromethane-acetone solution was evaporated to dryness on a N-Evap evaporator (Organoma- tion Assoc.) under a gentle stream of nitrogen using a water bath setting at 50°C. Special care was taken to avoid over- heating of the dry extract. The aflatoxin extract was then dissolved in a 100 /11 of benzene-acetonitrile (9:1) and the vial was sealed with a teflon lined screw cap. For samples extracts with high aflatoxin concentrations, the volume of the benzene-acetonitrile (9:1) was adjusted to give the 65 proper concentration of aflatoxins for densitometric analysis The vial containing the aflatoxins was shaken vigorously for about 1 minute on a vortex shaker before removing the sam- ples for analysis. Thin Layer Chromatography As shown in Figure 5,6 and 7 three different sizes of TLC plates were used to carry out the aflatoxin analysis: (1) 10x10 cm, (2) 10x20 cm, and (3) 20x20 cm. The 10x10 cm TLCplates (Figure 5) were made by cutting a precoated 20x20 cm silica gel plate (Sil-G-HR-ZS, Brinkman Instruments Inc.) into four equal parts. The TLC plates were cut using a glass cutter (Sargent-Welch, #S-39885). The 10x10 cm TLC plates were used for qualitative analysis of aflatoxins extracted from the raw tissues. The plates were scored and spotted as shown in Figure 5 A 20,ul sample of the aflatoxin extract was applied to the sample spot with a 25241 Syringe (Hamilton Co). Standards of 2.51, 0.88, 2.0 and 2.0 ng of aflatoxin B1, B2, M and M 1 respectively, were spotted on the plates as standards in 2: both directions of development. The plates were develOped in the first direction with chloroform-acetone (3:2) in an unlined and unequilibrated 1.5 liter beaker, which was tightly covered with aluminum foil. After the development in the first dimension was com- pleted, the plates were removed from.the beaker and dried under a hood for about 2 minutes. The plates were then 66 2nd direction —é>- .\ 1F standard 6 U '\ spots sample spot 0 5.9 E o If} H—* :4 1.5 cm 7 cm Figure 5 — Spotting and Scoring Pattern for Two-Dimension 10x10 cm TLC Plates. lst direction 67 lst direction 3 O K I a o :H J.) o o 3:. 6 standard :o 0 spots :o a O‘ N sample spot E . 0‘. O 0 Ln ;._,.,pt1, aihe—-———-—4H 1.5 cm 5 cm 3 cm Figure 6 — Spotting and Scoring Pattern for Two—Dimension 10x20 cm TLC Plates. 68 2nd direction )' f O s U 0 m \\ standard spots a 0 sample spot xi: 0 \ 0 l: t i—“l 2 cm. 12 cm 6 cm Figure 7 — Spotting and Scoring Pattern for Two-Dimensional 20x20 TLC Plates. lst direction 69 transferred to an oven with the temperature set at 50° C and dried for an additional 1 minute under a stream of nitrogen. After the plates were removed and allowed to cool for approx- imately 1 minute, they were developed in the second direction with anhydrous diethylether-methanol-water (90:8:2) as described earlier. After development was complete, the plates were removed, dried under a hood and then examined in a UV cabinet (Ultra-Violet Products, Inc.). The sample spots were compared with the reference standard spots in order to determine their chromatographic equivalence. The 10x20 cm TLC plates were obtained by cutting a 20x20 cm precoated TLC plate in two halves as shown in Fig- ure 6. These plates were used for quantitative analysis of aflatoxins extracted from raw tissues. The plates were scored and spotted as indicated in Figure 6 and as described earlier herein. The plates were deve10ped in the first direction with chloroform-acetone-isopropanol (85:10:5) in a sealed and unequilibrated tank. After the develoPment in the first direction was completed, the plates were removed from the tank and dried as before. After evaporation of the solvent, the plates were developed in the second direction with anhyé drous diethyletherdmethanol-water (95:4:1). After develop- ment in the second direction, the plates were dried 1 minute under a hood and prepared for densitometric analysis. The 20x20 cm precoated Silica gel plates were scored and spotted as shown in Figure 7. These plates were used 70 for quantitative analysis of aflatoxins extracted from the raw and cooked bellies, bacon and other cured and cooked tissues. The plates were developed in the first dimension with chloroform-acetone-isopropanol (87:10:3). After the development, the plates were dried as described earlier. The plates were then developed in the second dimension with anhydrous diethylether-methanol-water (95:4:1). They were then dried and prepared for densitometric analysis. Densitometric Analysis of Aflatoxins A double beam spectrodensitometer SD 3000-4 (Schoeffel Instruments) equipped with a 3380-A integrator (Hewlett- Packard) was used for quantifying the aflatoxin spots on the TLC plates. The plates were scored prior to spotting as shown in Figure 6 and 7 (Schoeffel Scoring Device - SDA 303), providing 10 mm strips. The average of three readings of the aflatoxin reference standards (spotted within the three strips parallel to the second direction of develoPment) was used for densitometric comparison in calculating the concen- trations of the sample spots. For the analysis of the stan- dards spots, the scanning beam was focused on each of the three stips and scanning was carried out. In scanning the sample spots, the plates were viewed under UV light, and each spot was localized within two pencil marks made on the silica gel layer. The marks were about 1 cm.apart and lo- cated approximately 3 mm ahead of the sample spot along the second dimension of development. The plates were then 71 placed on the plate carrier and with the beam of light set up at 540 nm (green light) so that the position of each spot on the plate was recorded by focusing the light beam within the two marks. Then each spot was scanned by driving paral- lel to the second dimension with the scanning beam. The spectrodensitometer was operated in the reflectance mode. For excitation, the monochromator was set to 365 nm and a secondary filter (430 nm band) was used to collect the emit— ted fluorescence around 425 nm. The secondary filter only permits the emitted or visible fluorescent light to pass into the phototube so that all ultraviolet light from the lamp is screened out. Aflatoxins B1, B2, M1 and M2 concentrations were cal- culated according to the following formula: «g/kg = (BxYxSxV)/(ZxXxW) were: B = Area of the aflatoxin peak in the sample spot, Y = Concentration of aflatoxin standard in qg/ml, S =.q1 of the aflatoxin standard, V = Dilution of sample extract in,ul, Z = Area of aflatoxin standard peak (average of three replications), X =l(l of sample extract spotted on the plate, and W = Grams of sample in the final extract. The weight of tissue represented in the final afla- toxin extract (the value W in the formula above) was calcu- lated based on the amount of water in the sample tissue, the 72 volume of extracting solution added to the tissue homoge- nate and the volume of filtrate collected in the two filtra- tion steps. For bacon and belly samples, the volume of fat in the tissue was considered since fat also is soluble in the solvents. For lean tissues, the fat content was not accounted for in the calculation. The moisture and fat con- tent of the tissues were determined by the A.O.A.C methods (1965). The sample weight in grams (W) represented in the final aflatoxin extract, was calculated according to the following formula: W= 235 ml X 325 m1 X initial weight (300m1+42ml+ml of water in sample) (20ml+150m1+235ml) of sample (g) Using the formula above, values of 235 and 325 m1 are the volumes of the first and second filtrate, respectively. The other values refer to the volumes of extracting solutions added to the tissue homogenate. Analysis of Aflatoxins in the Feed Analysis of the feed was carried out according to a modification of the A.O.A.C. method (1965). A 50 g sample of feed was weighed into a 500 m1 Erlenmeyer flask. Then, 25 ml water, 25 g of diatomaceous earth and 250 m1 of chloro- form were added. The stOpper was secured with masking tape and the flask was shaken for 30 minutes on a wrist action shaker (Burrel Corp.). The material was then filtered through prefolded filter paper (Whatman 2 V) and the first 73 50 ml of filtrate were collected, dried and transferred to a 10 g silica gel column as described earlier herein. The aflatoxins were eluted from the column, spotted in 20x20 cm TLC plates and quantified as described before for analysis of aflatoxins in tissue samples. Analysis of Aflatoxins from Drip of Cooked Meat The analysis of drip from the cooked tissues was car- ried out using a modification of the method of Trucksess and Stoloff (1979). The drip was weighed and transferred to a 500 ml Erlenmeyer flask. Then, 200 m1 acetone plus 40 ml NaCl-citric acid solution was added. The flask stOpper was secured with masking tape, and the flask was shaken for 30 minutes using a wrist action shaker. Then 150 m1 of water, 20 ml of lead acetate solution, 10 g of ammonium sulfate and 10 g of diatomaceous earth were added to the flask.. The solution was stirred for about 1 minute using a magnetic stirring device and then filtered through a fast filtering filter paper. A total of 325 ml of the filtrate was trans- ferred to a 500 ml separatory funnel and the solution was defated as before. The chloroform extracts were purified by silica gel column chromatography as described earlier herein. The final extract was spotted, developed and quantified using 20x20 cm TLC plates and the same solvent system that was used for analysis of cooked tissues. 74 Confirmatory Tests for Aflatoxins Aflatoxin B1 Twenty.ul of sample extract were applied about 4 cm from both edges in the left corner of a precoated TLC plate (20x20 cm Sil-G-HR-25, Brinkman Instruments, Inc.). Approx- imately 2.5, 0.5, 2.0 and 2.0 ng of aflatoxins B1, B2 and M1 and M2 standards, respectively, were spotted in the same lo- cation on right corner of the plates. Then the plates were developed in a closed, unlined and unequilibrated tank using anhydrous diethylether—methanol-water (96:3:1). After devel- opment, the plates were air dried under a hood for about 2 minutes and then dried in a 50°C oven under a stream of ni- trogen for about a minute. The spot corresponding to aflatoxianl was identified by comparison with the aflatoxin B1 standard. Then it was marked in the silica gel on the left with a pencil along the direction of the development. Another pencil mark was made about 1 cm apart to the left of the first. The second mark was used as a guide to apply about 2.5 ng of aflatoxin Bl standard close to the aflatoxin B1 sample spot. Then 2;Il of trifluoroacetic acid (TFA): chloroform (1:1) were applied to both of the aflatoxin B1 spots. The plate was allowed to stand in the dark for about 5 minutes at room temperature. Then the plate was dried in oven for 10 minutes at 45°C. After cooling the plate in the dark at room temperature, another 2.5 ng of aflatoxin B1 standard was applied about 75 1 cm to the left of the second pencil mark. The plate was developed in the second direction with chloroform-acetone- isopropanol (87:10:3) the same way as described before. After the plate was deve10ped and dried, the chromatogram was examined for the formation of aflatoxin B2a’ which has a lower Rf than the unreacted aflatoxin B1 standard. The chro- matographic equivalence of the sample and the aflatoxin B1 standard spot after treatment with TFA was used as a confir- matory test for identifying aflatoxin B1' Aflatoxin M1 The TLC plate was spotted, developed in the first di- rection and dried as described earlier herein in order to carry out the aflatoxin B1 confirmatory test. Then the afla- toxin M1 spot in the sample was marked in the silica gel on the left with a pencil along the direction of the develop- ment. Another pencil mark was made about 3 cm apart to the right of the first and close to the aflatoxin M2 spot. The second mark was used as a guide for applying about 2.0 ng of aflatoxin M1 standard. TWo,ul of TFA: chloroform (1:1) were then applied to both the sample spot and the aflatoxin M1 standard. The plate was allowed to stand in the dark for about 5 minutes at room.temperature. Then the plate was dried in a chromatographic oven for 5 minutes at 75°C. The plate was then cooled in the dark at room temperature and another 2.0 ng of aflatoxin M standard was spotted about 1 l cm.to the right of the second pencil mark. Then the plate 76 was developed perpendicular to the first direction using chloroform: acetone: isopropanol (85:10:5) for development. After develoPment, the plate was dried and examined under UV light for the formation of the aflatoxin M1 derivative in order to ascertain if the Rf was lower than that of the un- reacted aflatoxin M1 standard. The chromatographic equiv- alence of the sample and the aflatoxin M1 standard spot after treatment with TFA was used as a confirmatory test for the identity of aflatoxin M1. General Confirmatory Test for Aflatoxins This technique offers additional confirmation for the presence of aflatoxins at low levels (Przybylski, 1975). The technique is as follows: after development of the TLC plate and identification of the spots under UV light, the plate was sprayed with 25% sulfuric acid (v/v). Then the plate was dried in a chromatographic oven at 45°C under a stream of nitrogen. Changes in the characteristic fluores- cence of aflatoxins Bl’ B2, M1 and M2 from.blue to yellow after the H2804 treatment was used as an additional confir- matory test for the presence of aflatoxins. Three-Dimensional Chromatography This test is based on comparison of the chromatogra- phic mobilities of aflatoxins from.the sample extract with reference to authentic standards after three-dimensional chromatography using three different solvent systems. The 77 aflatoxin spot in the sample was eluted from the plate and spotted along with the aflatoxin standard in the same place in a 10x10 cm TLC plate. The presence of only one spot after three-dimensional chromatography was used as an additional confirmatory test for the identity of the aflatoxins in the sample extract. The aflatoxin spot in the sample was eluted from the plate using the following procedure: A ball of glass wool was applied into a 5 3/4 inches long glass disposable pipet (Scientific Products). Then a layer of about 0.5 cm of anhy- drous sodium sulfate was added. The tip of the glass pipet was then attached to rubber vacuum tubing connected to a water aspirator. The spot on the TLC plate was visualized with a UV cabinet and the aflatoxin spot to be extracted was marked on the silica gel layer using four pencil marks to define its boundary. Then about 2 [41 of water were applied to the aflatoxin spot to displace the aflatoxins from the silica gel adsorbent. The silica gel layer containing the aflatoxin was then removed by scraping it from the plate with the edge of the prepared glass pipet. The vacuum gener- ated inside the pipet was adequate to aspirate and collect the flaked silica gel spot. Once the spot area was removed from.the silica gel plate, the glass pipet was disconnected from the aspirator and clamped to a metal support. The aflatoxin was then eluted from the silica gel using 3 m1 of acetone, and the eluate was collected in a 1 gram vial. The acetone was then evaporated under a gentle stream of 78 nitrogen as described earlier hearin. The aflatoxin extract was dissolved in 40,u1 of chloroform. The entire chloroform extract was then spotted at a point located about 1.5 cm from both edges in the left corner of a 10x10 cm silica gel plate. About 2 ng of the aflatoxin standard was then super— imposed over the sample spot. The plates were then devel- oped in 1.5 liter beaker tightly covered with aluminum foil. After deve10pment in one direction, the plate was dried and then developed perpendicular to the first direction as ex- plained earlier herein. The following solvent systems were sequentially used for three dimensional development: (1) chloroform-acetone (85:15); (2) anhydrous diethylether-meth- anol-water (90:8:2); and (3) chloroform-acetone-iSOprOpanol (85:10:5). This technique was specially develOped for confirmatory identification of aflatoxin B2 and M2, which can not be iden- tified using chemical reagents to form derivatives. It can also be used as a confirmatory test for aflatoxin B1 and M1, using the TFA procedure if the background in the sample ex- tract is too intense to permit good visualization of the spots. In this case after quantitation of the sample spots, the B1 or M1 spot was removed from the plate and respotted on a 20x20 cm TLC plate as described before. Then the cor- responding aflatoxin standard spot was applied clOse to the sample spot and followed by the TFA treatment, drying and development of the TLC plates as described herein in the confirmatory test for aflatoxins B1 and M1. 79 Preparation of Aflatoxin Reference Standards Aflatoxins B1, B2, M1, M2 and 32a used as reference standards were obtained from Applied Science Division. The aflatoxins B1 and B2 used in the preparation of the spiked diets were purchased from CAL Biochem. Aflatoxin reference standards were prepared according to the AOAC method (1975) and contained 0.501, 0.175, 0.4 and 0.4}xg/m1 of aflatoxins B1, B2, M and M respectively. 1 2’ A solvent mixture of benzene-acetonitrils (98:2) was used as the solvent for aflatoxins B1 and B2, while Chloroform.was utilized as the solvent for aflatoxins M1 and M2. The afla- toxin standard solutions were stored at-20°C. Fat and Moisture Analysis Moisture Content The A.O.A.C. (1965) procedure for determining moisture was used. Five grams of tissue were accurately weighed to four decimal places into a previously dried and tared alumi- num dish (100°C for at least 1 hour). The sample plus the dish were then dried overnight for 18-24 hours in an air con- vection oven at 100°C. The dried sample was cooled in a des- iccator and weighed to four decimal places. Loss in weight was reported as moisture for each hundred grams of meat. Three replicates were run for each sample. 80 Fat Content The fat content was determined using the Goldfisch ex- traction method of the A.O.A.C. (1965). The same sample was used following moisture analysis. The aluminum dish containing the dried meat sample was carefully folded into a porous thimble and clipped into a Goldfisch apparatus. The fat was extracted with anhydrous diethylether for approximately 8 hours into a previously dried and tared beaker. The extract was then dried for 1 hour at 100°C in an air convection oven, cooled in a desiccator and weighed as before. The percent fat was calculated as grams of fat extracted from each one hundred grams of tissue. Three replicates were run per sample. Safety Procedures All glassware and vials in contact with aflatoxins were soaked either with 5-6% NaOCL (household bleach) or with sulfuric acid-dichromate solution (120 g Na20r207.2H20 + 1600 ml conc. H2804 and diluted to a volume of 3 liters with water) to destroy any residual aflatoxins. Plastic disposable gloves were worn routinely during all work with aflatoxins. Respirator masks were worn when mixing and han- dling the spiked rations. Surface work areas were routinely scanned with a UV lamp and any contaminated areas were treat- ed by washing thoroughly with 5.6% NaOCl solution. All TLC plates used in aflatoxin analysis were thoroughly soaked in 81 NaOCl solution before discarding. Filter papers and tissue residues resulting from aflatoxin analysis were thoroughly soaked with concentrated ammonium.hydroxide solution over- night before discarding. These waste materials, after treat- ment with ammonia, were collected in plastic bags and placed inside tightly closed containers and labeled properly until removed by the MSU Animal Waste Disposal Unit. All work in- volving scraping of the plates was done under a hood. Simi- larly any work involving the use of toxic solvents, sudh as benzene, chloroform and acetonitrile, were also performed under the hood. This involved preparation of silica gel columns, spotting, develoPment and drying of the TLC plates. RESULTS AND DISCUSSION Feedinngrial The response of the animals to aflatoxins was deter- mined by growth, feed consumption and organ weights. Afla- toxin intake by the pigs was expressed as the amount of the toxin ingested per unit of body weight per day, which is the daily dosage rate (DR). Armbrecht gt gt. (1971) concluded that DR gives the best measure of the response of the exposed animals to aflatoxins. Except for the feed of the control pigs on trials 1 and 2, the experimental conditions for both trials 1 and 2 were identical. The feed of the control pigs on trial 1 was naturally contaminated with 20 and 31 ppb of aflatoxins Bl and B2, respectively, while in trial 2 the feed was free of aflatoxins. In trial 1 the low level of contamination in the basal diet turned out to be a fortuitous circumstance. It was found that there were no significant differences between the weight gains and organ weights of the control pigs and those fed the aflatoxin spiked diet. This is in contrast to the comparison between the aflatoxin free controls and pigs 82 83 fed a spiked diet in trial 2. The control pigs in trial 1 exhibited depressed growth and on the average gained 40 per- cent less weight than the controls in trial 2 over the same feeding period (Table 4). Furthermore, the controls in trial 1 also showed an average of 24 percent reduction in feed intake as compared to the controls in trial 2 (Table 5). The data from Furtado gt gt. (1979) were also in contrast to those in trial 1 as they found significative differences in gains between the controls and pigs fed aflatoxins. Results indicate that even at the low levels of afla- toxins found in the naturally contaminated diet, deposition of small amounts of aflatoxins occurred in the liver and kidneys of the control pigs (Table 8). Although no visual lesions were observed in the liver and kidneys of control pigs in trial 1, results suggest that exposure to levels as low as 20 to 31 ppb of aflatoxins B1 and B2, respectively, was adequate to cause adverse effects on the performance of the pigs. 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(g) Dried Wt. (g) Moisture CZ) Raw Loin 1 4.9382 1.1392 76.9 2 4.8883 1.1483 76.5 3 5.2947 1.2409 76.6 Mean - - 76.7 Cooked Loin 1 5.9367 2.5931 56.3 2 4.7780 2.2111 53.7 3 4.7032 1.9536 58.5 Mean - - 56.2 TABLE 20 - Aflatoxin Levels (neg/kg) Expressed as Dry Weight Basis Found in Raw Loin Tissues and Raw-Cooked loin Pig No.a Raw Raw-Cookedb B]. B]. 32 0.85 0.58 0.75 l 0.80 0.54 0.70 . 0.56 0.73 1.07 1.60 0.85 1.20 2 1.18 1.78 0.80 1.25 1.13 1.69 0.83 1.23 1.91 1.61 1.41 0.82 3 1.72 1.25 1.33 1.00 1.82 1.43 1.37 0.91 0 77 0.60 0.75 0.58 4 O 67 0.44 0.64 0.50 0 72 0.52 0.70 0.54 aEach sample represents a loin from a different pig fed the aflatoxin-spiked diet at zero day withdrawal time. bThe sample was cooked by broiling as described in the experimental section. .SOHuoom HmueoEHHmmxo mnu cH nmnHHommm mm nommmooum 003 mHmEmm one Q .mBHu HmsmupnuHB mhmp oumn um uoHv momem1aonumHmm man now me quHmMMHn m Eonw an: m mucommummu oHdamm norm m 133 E. E F1. 0 E II: m . Em . .0111; . 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