Dietary factors and aflatoxin toxicity: I. comparison of the effect of two diets supplemented with aflatoxin B1 upon two different strains of rats

Previous studies in this laboratory with rats fed low levels of aflatoxin as a component of peanut discards, suggested either a strain tolerance for aflatoxin or a possible protective factor present in the diets used. In this study, two strains of rats, the Charles-River strain and the former USC strain, were used to test the effect of 1.7 ppm purified aflatoxin B₁ included for 3 months in two different diets; one previously used in this laboratory and one used by other investigators in aflatoxin studies. After an experimental period of either 12 or 18 months, growth, mortality, gross pathology, and organ wt were measured, and histopathological examination and biochemical analyses were performed. Plasma and liver cholesterol levels, total liver lipids, and fatty acids in the various lipid fractions of plasma, liver, and liver tumor lipids were measured. Both strains of rats proved to be susceptible to aflatoxin toxicity at this level as manifested by the appearance of hepatomas; however, liver involvement was more extensive in the Charles-River rats. The diet used by other investigators produced symptoms similar to those observed as a result of essential fatty acid deficiency and also affected the response to aflatoxin through an aggravation of symptoms, i.e. an inhibition of growth and increased size and severity of the liver tumors.     

Safety of nuts heat-processed in molten hexitols

A blend of hexitols, comprising 80 parts by weight of mannitol and 20 parts of sorbitol, has been used in place of a frying fat in roasting nuts. Even though physicochemical tests demonstrated no measurable deterioration of the hexitol components on continuing repetitive use, biological studies were carried out to establish the safety of the end product. The latter was the novel dry roasted peanut product; the control consisted of conventionally air roasted peanuts. Both products were ground to peanut butter consistency and then stabilized with added hydrogenated peanut oil to facilitate the feeding programs. Male and female rats were maintained on the two peanut products for four generations. It was found that rats subsisting on diets containing the regular peanut butter increased in weight at a somewhat greater rate than did those on the diet containing the special peanut product. Efficiency of food utilization was not adversely affected and overall weight gains were satisfactory. All rats appeared normal and healthy throughout the experiment. There was no morbidity and no diarrhea. Breeding performance was slightly better among those rats on the special peanut product diet. No pathology was observed at autopsy and there were no differences in organ weights among the rats on the two diets. Total liver lipid and liver cholesterol levels were somewhat higher among the control peanut butter-fed rats, as compared to those on the “special” peanut butter diet. On the basis of (a) published related studies involving biological evaluations of the control peanut butter diet vs. the same adequate diet but containing no peanut components and (b) the present findings, it is concluded that no unknown factors of a toxic nature are present in peanuts roasted in the molten hexitol blend.     

Aflatoxin content of peanut hulls

The degree of aflatoxin contamination in peanut hulls was determined by analyzing inoculated hand-shelled hulls and hulls from peanuts known to contain aflatoxin. Hulls adjusted to 20% moisture, inoculated withAspergillus flavus, and incubated 7 days at 25 C supported growth ofA. flavus but not aflatoxin production. Peanuts from 20 selected Segregation III (visibleA. flavus) lots contained 13–353 ppb of aflatoxin. The machine-shelled hulls from these lots were analyzed and 3 lots contained no detectable aflatoxin, 13 lots contained 4–88 ppb and 4 lots contained >116 ppb. Aflatoxin concentrations of 53–87 ppb were detected in hulls when peanuts containing relatively high levels of aflatoxin (up to 26.8 ppm in damaged kernels) were carefully machine-shelled. Hulls from the same samples obtained by hand-shelling contained no detectable aflatoxin. When machine-shelled hulls were screened through successively smaller screens, the aflatoxin concentration of the smallest fraction (<3.18 mm) was always highest and indicated that small peanut kernels and peanut parts in the hulls actually contained the aflatoxin. Separating hulls over a 4.76 mm round-hole screen appeared to provide a means of removal of most aflatoxin in peanut hulls. No aflatoxin was found in hulls from uncontaminated peanuts.     

Influence of aflatoxin B1 on growth of rats under various nutritional conditions

The effect of the addition of aflatoxin B1 was studied in 48 male, Sprague-Dawley rats weaned at 21 days of age. The animals of the experimental group received one of the following diets to which aflatoxin B1 was added (5 mg/kg ration), a protein-free diet, the regional basic diet of Northeast Brazil (RBD) containing 9.07% protein, and commercial casein diets (at 10 and 20%). The control groups were fed the same diets without aflatoxin B1. To detect possible body alterations, the body weight and food intake, as well as protein and aflatoxin B1 intakes were recorded. Significant alterations were detected in the experimental groups, especially in the protein-depleted animals, and in those fed the RBD and 10% commercial casein diets, when compared to their controls.     

Aflatoxin effects in livestock

Feeding trials were conducted with swine, beef cattle, dairy cattle and poultry to determine adverse effects, if any, of graded levels of aflatoxins in rations. In addition, samples of meat, eggs and milk from these animals were analyzed chemically to determine if aflatoxin was transmitted into these products. In growing-fattening swine, no evidence of toxic effects was observed when the aflatoxin level fed was 233 ppb or less. In a swine reproduction experiment, no adverse effects were detected in pigs produced from sows fed 450 ppb aflatoxin. No toxic effects were observed at levels of 300 ppb or lower in cross-bred beef steers fed aflatoxin rations for 4.5 months. Using recognized chemical methods, we detected no aflatoxin in meat from swine and cattle fed rations containing 800 and 1000 ppb of aflatoxin, respectively. In dairy cows, weekly intakes of 67 to 200 mg of aflatoxin B₁ per cow produced 70 to 154 ppb aflatoxin M₁ in lyophilized milk. Rapid disappearance of aflatoxin M₁ in the milk took place after withdrawal of aflatoxin from the ration. No adverse effects were discernible in broilers fed from one day to eight weeks of age a ration containing 400 ppb aflatoxin. Lyophilized meat from broilers fed 1600 ppb aflatoxin for eight weeks contained no detectable aflatoxin. Striking differences in aflatoxin susceptibility were observed in 17 different breeds and strains of poultry and game birds fed from two to six weeks of age a ration of 800 ppb aflatoxin B₁. New Hampshire chicks and turkey poults were highly susceptible to aflatoxin in contrast to the resistance of Barred Rock and Australop chickens and guinea fowl. Hybrid chicks from a New Hampshire-White Leghorn cross were highly resistant to aflatoxin. Eggs and meat from White Leghorn hens fed a ration containing 2700 ppb aflatoxin contained no detectable aflatoxin. One of 21 papers presented at the Symposium, “Oilseed Processors Challenged by World Protein Need,” ISF-AOCS World Congress, Chicago, September 1970. W. Utiliz. Res. Dev. Div., ARS, USDA.     

Effect of varying levels of dietary protein on tumor development and lipid metabolism in rats exposed to aflatoxin

Reports in the literature concerning the relationship of protein nutrition to aflatoxicosis are contradictory. In an attempt to elucidate this relationship more clearly, we have examined the effects of low, normal, and high protein-containing diets on tumor incidence and development, as well as on several biochemical indices, in rats which have been exposed to low levels of aflatoxin in a “chronic” rather than “acute” situation. In our study, male weanling rats were place for 3 months on otherwise adequate diets containing either 8, 22, or 30% casein with and without aflatoxin B₁ at 1.7 ppm. Half of the animals in each group received diets which were further supplemented with the amino acid, cystine, at 0.6% of the diet. (Sulfur-containing amino acids are the most limiting amino acids in casein, and the addition of cystine to the diet serves to improve the biological quality of the protein source.) After 3 months the animals were fed control diets without aflatoxin until they were killed at 1 year. Weight gain was markedly decreased and liver weights increased in response to aflatoxin in all groups except those on the low protein diets, where aflatoxin had no effect on these protein diets, where aflatoxin had no effect on these indices. No tumors were found in the livers of rats fed the low protein, aflatoxin-supplemented diet. In the other groups, the severity of the liver involvement increased progressively with increased protein levels in the diet. When cystine was included in the diet, tumors were observed also in the animals fed the low protein diet; furthermore, the livers of those animals on “normal” and high protein diets were much more severely involved than were the livers of animals on non-cystine supplemented diets. Plasma cholesterol levels were increased in response to aflatoxin when the diets containing 22 and 30% protein were fed and when cystine was included in the 8% protein diet. Liver cholesterol levels were increased in response to aflatoxin in all groups except in those receiving the low protein diets. Among these latter animals, aflatoxin administration had no effect on liver cholesterol values. Changes as a result of aflatoxin administration were also observed in the fatty acid composition of sterol esters, triglycerides, and phospholipids of liver and tumor tissue.     

Destruction of aflatoxins in peanut protein isolates by sodium hypochlorite

Sodium hypochlorite has been tested for destruction of aflatoxins during the preparation of peanut protein isolates from raw peanuts and defatted peanut meal. The treatments were evaluated by determination of the aflatoxins in the products by thin layer chromatography. Effects of sodium hypochlorite concentration, reaction pH, temperature, and time were studied. Results show that both the sodium hypochlorite concentration and pH are important factors in reducing the concentration of aflatoxins in the protein isolates to nondetectable levels. The treatment with 0.4% sodium hypochlorite at pH 8 produced protein isolates with trace amounts of aflatoxins B₁ and B₂ from ground raw peanuts containing 725 ppb aflatoxin B₁ and 148 ppb aflatoxin B₂, whereas untreated protein isolates contained 384 ppb aflatoxin B₁ and 76 ppb aflatoxin B₂. At pH 9, 0.3% sodium hypochlorite reduced the aflatoxin B₁ content in the protein isolates from 300 ppb to below detectable quantities and the aflatoxin B₂ content from 52 ppb to 2 ppb. Similar results were obtained at pH 10 for 0.3% sodium hypochlorite concentration. In the case of defatted peanut meal which contained 136 ppb aflatoxin B₁ and 36 ppb aflatoxin B₂, 0.25% sodium hypochlorite concentration at pH 8 (0.20% at pH 9; 0.15% at pH 10) reduced both the aflatoxin B₁ and B₂ contents to below detectable quantities in protein isolates as compared to aflatoxin levels of ca. 75 ppb B₁ and 17 ppb B₂ in the untreated protein isolates. Reaction temperature and time did not affect the destruction of aflatoxins significantly.     

Effect of sterculic acid upon aflatoxicosis in rats fed diets containing saturated and unsaturated fat

Investigations on trout have shown that the cyclopropenoid fatty acids, which occur naturally in small amounts in unrefined cottonseed oil, may act as powerful cocarcinogens when fed in conjunction with aflatoxin. Attempts at confirming these findings in mammals, i.e. rats, have been inconclusive. In this study, the effects of sterculic acid and aflatoxin upon lipid metabolism and tumor formation in male rats have been examined using basal diets containing either saturated or unsaturated fat to which the following additions were made: (A) basal diet (no supplements); (B) aflatoxin B₁ at 1.7 ppm; (C) sterculic acid at 210 ppm; and (D) aflatoxin B₁ at 1.7 ppm, plus sterculic acid at 210 ppm. The rats consumed these diets for 3 months and, thereafter, were fed the unsupplemented basal diet until sacrifice 9 months later. Growth was depressed in rats in groups B, C, and D, but no synergistic inhibition was observed, regardless of the fat source. Liver wt doubled in response to aflatoxin; however, only when the diet contained unsaturated fat did sterculic acid, in combination with aflatoxin, exaggerate the increase in liver wt (a reflection of the more severe liver pathology observed in these rats). In the animals fed the saturated fat diet, aflatoxin administration to animals fed the control or sterculic acid supplemented diets resulted in marked increases in plasma cholesterol levels; the unsaturated fat diets, supplemented with aflatoxin, evoked a slight increase in plasma cholesterol content which was nullified by sterculic acid supplementation.     

Effect of dietary fat upon aflatoxicosis in rats fed torula yeast containing diet

This study was designed to study the possible interrelationships between Torula yeast, vitamin E, and the dietary fat source on aflatoxin-induced tumors. Rats were fed Torula yeast-containing basal diets which included 1.7 ppm aflatoxin B₁ with either lard, corn oil or no fat, and with or without vitamin E supplements for 3 months. Thereafter, the respective diets without aflatoxin were fed for ca. 9 months. Animals receiving the vitamin E-deficient diets had a high mortality. Although the vitamin E-deficient, aflatoxin-treated rats had lower wt gains than did the vitamin E-deficient controls, they lived twice as long. In addition, regardless of the dietary fat source, the kidneys and adrenals of these vitamin E-deficient, aflatoxin-supplemented rats were found to be significantly heavier than the controls, and plasma cholesterol levels were elevated. Increased amounts of liver lipid were observed in response to aflatoxin in both corn oil-fed and fat-deficient rats. No such differences were observed in the responses of the vitamin E-supplemented groups to aflatoxin. On the corn oil diet, aflatoxin administration resulted in an increased deposition of polyunsaturated fatty acids in cholesteryl ester and phospholipid fractions in livers of vitamin E-deficient rats and the phospholipid fraction of vitamin E-sufficient rats. The vitamin E-deficient rats exhibited necrosis of the liver, which was alleviated when aflatoxin was included in the diet, and calcification of the kidneys, which was potentiated by the dietary aflatoxin. No tumors were observed in these animals. In animals maintained on vitamin E-sufficient diets for 1 year, growth was depressed as a result of aflatoxin administration with the greatest depression occurring in the group fed corn oil. Spleen wt were decreased in all groups given aflatoxin. However, there were no changes in either plasma or liver cholesterol or total liver lipids which could be attributed to aflatoxin administration. When aflatoxin was fed with lard, the cholesteryl ester, triglyceride, and free fatty acid fractions of plasma had decreased amounts of the C20:4 acid. In the cholesteryl ester fraction only, this change was accompanied by increased levels of C16:0, C18:0, and C18:1 acids. In the liver phospholipids, there were increased levels of mono- and polyunsaturated fatty acids and decreases in the saturated fatty acids. All of the animals receiving aflatoxin exhibited severe necrosis and tumor formation in the kidneys; the animals fed lard had the highest level of involvement and those in the fat-free group the least. Liver pathology was the least marked among the rats fed the fat-free diet. Since aflatoxin-induced tumors are rich in lipids, the fat-free diet may be protective to the animal.     

Dietary factors and aflatoxin toxicity: II. effect of fat source upon aflatoxicosis in rats

Previous studies in this laboratory have indicated that the tumorigenic and biochemical response of rats to aflatoxin may be affected by diet. To clarify the possible importance of the fat component of the diet in altering these biochemical responses a cross-over experiment was devised in which peanut oil and lard were reversed in two diets containing different protein sources and vitamin mixtures. It was found that more concentrated doses of aflatoxin administered for a shorter period of time had a more inhibitory effect upon growth, and liver pathology also appeared to be worse than when smaller doses were given for longer periods of time. There were slight differences in pathology due to the basal diet among peanut oilfed rats, and large differences when the fat component was lard. Plasma cholesterol levels were elevated as a result of the aflatoxin administration, regardless of the diet, and liver cholesterol levels were elevated in response to aflatoxin when diets containing the more varied protein source were fed. In all fractions of plasma fatty acids, fatty acid patterns similar to those observed in marginal essential fatty acid deficiency were seen when lard was fed. The observations that the more restricted protein diet or some component of it, reacts with lard to produce signs similar to those typical of essential fatty acid deficiency and that this regimen produces the most severe liver pathology further establishes the importance of the diet as a means of modifying the response of the organism to aflatoxin.     

Effect of roasting oil composition on the stability of roasted high-oleic peanuts

Off-flavor due to lipid degradation is an important factor in the shelf life of peanut products. The use of recently developed peanuts with high-oleic acid/linoleic acid (O/L) ratio has the potential to significantly extend the shelf life of roasted peanuts. To determine the full potential for shelf-life improvement of oil-roasted high-O/L peanuts, a study was conducted to examine the effects of roasting high-O/L peanuts (O/L=30) in high-O/L (O/L=23.2) or conventional (O/L=1.5) peanut oil. Peanuts were roasted at 177°C to Hunter L values of 49±1. Roasted peanuts were stored at 30°C for 20 wk. Samples were taken at regular intervals to determine PV, oxidative stability index (OSI), moisture content, and water activity. The O/L ratio of high-O/L roasted peanuts was 27.9 vs. 13.6 for the conventional oil-roasted peanuts. After 20 wk of storage, PV of conventional oil-roasted peanuts was 10.8 compared to 5.3 for the high-O/L-roasted peanuts. OSI values were 88.5 and 52.4 immediately after roasting for the high-O/L-roasted vs. conventional oil-roasted peanuts. OSI for both decreased, but differences remained similar throughout the storage period. Shelf life of high-O/L peanuts decreased when roasted in conventional O/L-peanut oil vs. high-O/L peanut oil.     

Food uses of peanut protein

Approximately 19 million metric tons of peanuts (Arachis lypogae L.) are harvested annually, and contribute over 3.5 million tons to the world’s protein pool for food and feed uses. Peanut is the world’s fourth most important source of edible vegetable oil and the third most important source of vegetable protein feed meal. About 70% of the U.S. Crop is consumed domestically or exported as peanut kernels, peanut butter, and confections. Crushing is limited primarily to culls and kernels containing aflatoxin; and to stabilize the market. However, in countries such as India, Senegal, Brazil and Argentina, 75 to nearly 100% of the crop is crushed or exported for use as oil and livestock meal. The peanut is perhaps the world’s most widely researched food protein oilseed. Advantages over other oilseeds include relatively bland flavor, minor color problems, and minimal preparation requirements. Products in use throughout the world include boiled peanuts, roasted full-fat or partially defatted peanuts, peanut butters, grits and flours (full-fat or defatted), defatted peanuts, protein concentrates, and protein isolates. Compounded food applications include fortified breads and bakery products, snacks, meat products, extended milks, cheese and curd type products, and various mass-feeding foods in developing countries. Challenges encountered in peanut utilization include improvement of flavor levels and stability, identification of nutritional adequacy and fortification requirements, elimination of antinutritional factors, development of new products and improved processes, and elimination of aflatoxin problems.     

Effect of dietarytrans fatty acids on microsomal enzymes and membranes

Three groups of rats were maintained on diets containing different proportions oftrans fatty acids (0, 18.3 or 36.6% of the total fatty acids) for eight weeks. No differences in body weight were observed among the three groups, but the fat cell size, determined in epididymal fat, differed significantly between the controls and the rats fed diets containingtrans fatty acids. The supernatant obtained by centrifuging homogenates of liver from the rats at 9000×g (S-9 fraction) was used as an activator in a bacterial test for mutagenicity of 2-aminofluorene and aflatoxin B₁ usingSalmonella typhimurium strains TA 98 and TA 100, respectively. The mutagenicities of 2-aminofluorene in strain TA 98 and of aflatoxin B₁ in strain TA 100 were significantly lower with the liver S-9 fraction from rats fed a diet containing 36.6%trans fatty acids than with the liver S-9 fraction from rats fed a control diet with notrans fatty acids.     

A method for determining kernel moisture content and aflatoxin concentrations in peanuts

A method was developed to determine kernel moisture content (KMC) and aflatoxin concentration in discrete peanut samples. Shelled peanuts were weighed to the nearest 0.01 g, and a water slurry was made by blending the peanuts for 2 min with 2.2 ml of water per g of peanuts. The slurry (10 g) was withdrawn and dried at 130°C for 3 h to determine KMC. Methanol was added to the remaining slurry and blended for an additional 1 min, and aflatoxins were quantitated with high-performance liquid chromatography. Comparison of the slurry method with an official peanut moisture method showed good agreement between the two over a range of moisture levels. Recovery of aflatoxin B₁ from spiked samples averaged 97% with an average coefficient of variation of 3.6%. The method enables determination of both KMC and aflatoxin content in peanut samples without degradation of aflatoxin that would occur when using the official moisture method.     

Rapid method for detecting aflatoxins in peanuts

The thin-layer chromatographic procedure for detecting and quantifying aflatoxin in peanuts is too time-consuming, costly, and difficult to be practical for use by untrained personnel. A method based on millicolumn chromatography was tested and found to be both rapid and simple. Columns are prepared by filling a length of 4-mm glass tubing with silica gel to a depth of about 4.5 cm. The millicolumn is developed in a chloro-form-methanol extract of a peanut sample. If aflatoxin is present, a blue fluorescent band at the lower end of the column is observed when the column is exposed to long-wave ultraviolet radiation. Sensitivity is in the order of 5 ppb and an assay can be completed in 15–25 min. Some degree of quantification is possible by comparison with columns developed in extracts with known aflatoxin contents.     

Aflatoxin control program for Peanuts

Under provisions of a USDA Marketing Agreement, an aflatoxin control program for peanuts produced in the United States is administered by the Peanut Administrative Committee composed of peanut growers and shellers. Regulations of this committee contain provisions about the quality of peanuts acquired from farmers, storage of unshelled peanuts, aflatoxin testing, quality and disposition of processed lots, and indemnification of handlers for losses caused by lots which test over 25 parts-perbillion aflatoxin. Effects of the control program on aflatoxin concentrations in peanut products are discussed. Paper number 4978 of the Journal Series of the North Carolina Agricultural Experiment Station, Raleigh, NC 27607.     

Influence of medium-chain triglycerides on lipid metabolism in the rat

Lipid metabolism was studied in rats fed diets containing corn oil, coconut oil, or medium-chain triglyceride (MCT), a glyceride mixture containing fatty acids of 8 and 10 carbons in length. The ingestion of MCT-supplemented, cholesterolfree diets depressed plasma and liver total lipids and cholesterol as compared with corn oil-supplemented diets. In rats fed cholesterol-containing diets, plasma cholesterol levels were not influenced by dietary MCT, but liver cholesterol levels were significantly lower than in animals fed corn oil. In vitro cholesterol synthesis from acetate-1-¹⁴C was lower in liver slices of rats that consumed MCT than in similar preparations from corn oil-fed rats. Studies of fatty acid carboxyl labeling from acetate-1-¹⁴C and the conversion of palmitate-1-¹⁴C to C₁₈ acids by liver slices showed that chain-lengthening activity is greater in the liver tissue of rats fed MCT than in the liver of animals fed corn oil. The hepatic fatty acid desaturation mechanisms, evaluated by measuring the conversion of stearate-2-¹⁴C to oleate, was also enhanced by feeding MCT. Adipose tissue of rats fed MCT converts acetate-1-¹⁴C to fatty acids at a much faster rate than does tissue from animals fed corn oil. Evidence is presented to show that the enhanced incorporation of acetate into fatty acids by the adipose tissue of rats fed MCT represents de novo synthesis of fatty acids and not chain-lengthening activity. Data are also presented on the fatty acid composition of plasma, liver, and adipose tissue lipids of rats fed the different fats under study.     

A fluorometric rapid screen method for aflatoxin in peanuts

Peanuts were screened for aflatoxin using a rapid, inexpensive fluorometric method. Peanuts were ground and extracted with methanol, and the extract was treated with acidified zinc-acetate-sodium chloride solution, filtered, and diluted with water. Fluorescence of the extracts was compared with that from aflatoxin-free control peanuts. Test samples (160) of several varieties and grades of sources and were screened for the presence of aflatoxin. One hundred thirty-five samples (84%) were identified by this method as aflatoxin positive (15 ppb+) or aflatoxin negative (<15 ppb). Although 22 samples (13.6%) were incorrectly labeled as aflatoxin positive, most of these showed evidence of the presence of mold metabolites other than aflatoxin. Three samples (1.8%) were incorrectly labeled as aflatoxin negative when they actually contained 20, 33, and 34 ppb aflatoxin.     

Absence of aflatoxin from refined vegetable oils

The present investigation is the first definitive study of the fate of the aflatoxins in vegetable oils undergoing processing. Crude oils, obtained by solvent extraction or by hydraulic pressing of ground moldy peanuts (not suitable for human consumption), contained only small fractions of the aflatoxin originally present in the peanuts; the meals retained the bulk of the aflatoxin. Conventional alkali refining and washing of the oils reduced aflatoxin content to a range of 10 to 14 ppb. The subsequent bleaching operation essentially eliminated aflatoxin from the oils; the concentrations were now less than 1 ppb. The above results were confirmed using corn oils obtained from corn germ deliberately contaminated in the laboratory withAspergillus flavus. The nonfluorescing forms of aflatoxins, capable of being produced during the alkali refining operations, are also absent from the refined vegetable oils; these aflatoxin derivatives are readily converted to their original form on acidification and thereby measurable by fluorescence, if present. Presented in part at the AOCS Meeting, Los Angeles, April 1966.     

Nutritive value of heated vegetable oils

Peanut, sesame and coconut oils were heated at 270C for 8 hr, in an open iron pan. These fats were fed to albino rats at 15% level in otherwise adequate diets. All rats fed heated fats showed a growth depression. Livers of rats receiving heated oil were congested and showed extensive periportal fatty infiltration. Rats on heated peanut oil showed i) reduced B-vitamin storage in the liver, ii) increased glucose and cholesterol levels in the blood and iii) a disruption in the digestion and absorption of carbohydrates.