The clinical relevance of lipid transfer protein.

Despite a huge number of studies, many aspects of the lipid transfer protein (LTP) syndrome, the most frequent primary food allergy in Mediterranean countries, remain unclear. Its peculiar geographical distribution, along with the extreme variability of its clinical expression, makes this type of food allergy something unique in the panorama of IgE-mediated food-induced allergic reactions. This review article tried to summarize the current knowledge about the most important aspects of LTP sensitization and allergy, along with the importance of positive and negative co-factors in the clinical expression of the syndrome as well as the issues regarding the cross-reactivity between LTPs present in botanically related and unrelated foods. Further, the possible absence of the protein from some plant foods is discussed.

Toxicological studies on plant proteins: a review.

Nowadays, toxicological studies are contributing to human health more than ever. Reports on the toxicological studies of plant proteins, which are continuously growing in number in the literature, have been reviewed. Two important aspects are discussed: dietary safety evaluation, including toxicity tests and the maximum daily intake allowance, and the appropriate proportion in our daily diets of proteins from traditional foods and of new proteins from plant sources not traditionally employed as foods. Water hyacinth leaf proteins, sweet lupin proteins and canola proteins have not been shown to be toxic, although they are not traditionally employed as food proteins. These findings are very important for exploiting valuable new protein sources that are suitable for human or animal consumption and applicable to the food industry. Acutely toxic proteins, including lectins, ribosome-inactivating proteins, inhibitors of proteolytic enzymes and glycohydro-lases, have been isolated from plant materials and identified. Their toxicities and molecular characteristics have been described. The toxicity of proteins depends upon their specific native structures. Once they are denatured by appropriate treatment, such as heating, their toxicity can be reduced or even eliminated. These findings indicate that raw materials that contain this kind of toxic protein are not edible. However, after proper processing, they may be suitable for human or animal consumption. Although the toxicities of type 2 ribosome-inactivating proteins reported by different authors vary, the maximum dosages are still trace amounts.

A 90-day toxicology study of transgenic lysine-rich maize grain (Y642) in Sprague-Dawley rats.

The gene for a lysine-rich protein (sb401) obtained from potatoes (Solanum berthaultii) was inserted into maize seed to produce Y642 transgenic maize. Compositional analysis of Y642 grain demonstrated that the concentrations of lysine and total protein were higher than those observed in maize grain from a near-isogenic non-genetically modified (non-GM) commercially available control quality protein maize (Nongda 108). The safety of Y642 maize grain was assessed by comparison of toxicology response variables in Sprague-Dawley (SD) rats consuming diets containing Y642 maize grain with those containing Nongda 108 maize grain. Maize grains from Y642 or Nongda 108 were incorporated into rodent diets at low (30%) or high concentrations (76%) and administered to SD rats (n=10/sex/group) for 90 days. An additional group of negative control group of rats (n=10/sex/group) were fed AIN93G diets. No adverse diet-related differences in body weights, feed consumption/utilization, clinical chemistry, hematology, absolute and relative organ weights were observed. Further, no differences in gross or microscopic pathology were observed between rats consuming diets with Y642 maize grain compared with rats consuming diets containing Nongda 108 maize grain. These results demonstrated that Y642 lysine-rich maize is as safe and nutritious as conventional quality protein maize.

Epigenetic and phenotypic changes result from a continuous pre and post natal dietary exposure to phytoestrogens in an experimental population of mice.

BACKGROUND: Developmental effects of exposure to endocrine disruptors can influence adult characters in mammals, but could also have evolutionary consequences. The aim of this study was to simulate an environmental exposure of an experimental population of mice to high amounts of nutritional phytoestrogens and to evaluate parameters of relevance for evolutionary change in the offspring. The effect of a continuous pre- and post-natal exposure to high levels of dietary isoflavones was evaluated on sexual maturity, morphometric parameters and DNA methylation status in mice. Adult mice male/female couples were fed ad libitum either with control diet (standard laboratory chow) or ISF diet (control diet plus a soy isoflavone extract at 2% (w/w) that contained the phytoestrogens genistein and daidzein). In the offspring we measured: i) the onset of vaginal opening (sexual maturation) in females, ii) weight and size in all pups at 7, 14, 21 and 42 days post-natal (dpn) and iii) DNA methylation patterns in skeletal alpha-actin (Acta1), estrogen receptor-alpha and c-fos in adults (42 dpn). RESULTS: Vaginal opening was advanced in female pups in the ISF group, from 31.6 +/- 0.75 dpn to 25.7 +/- 0.48. No differences in size or weight at ages 7, 14 or 21 dpn were detected between experimental groups. Nevertheless, at age 42 dpn reduced size and weight were observed in ISF pups, in addition to suppression of normal gender differences in weight seen in the control group (males heavier that females). Also, natural differences seen in DNA methylation at Acta1 promoter in the offspring originated in the control group were suppressed in the ISF group. Acta1 is known to be developmentally regulated and related to morphomotric features. CONCLUSION: This study demonstrates in mammals that individuals from a population subjected to a high consumption of isoflavones can show alterations in characters that may be of importance from an evolutionary perspective, such as epigenetic and morphometric characters or sexual maturation, a life history character.

Fatty liver induced by the addition of excess cystine to a soy-bean protein diet in rats.

1. The effects of excess cystine added to diets with casein, egg protein, soya-bean protein and wheat gluten as protein source on liver and serum lipids of rats were compared. 2. The addition of excess cystine to a soya-bean protein diet produced lipid accumulation in the liver. 3. The addition of excess cystine to casein, egg protein and soya-bean protein diets, but not a wheat gluten diet, increased serum cholesterol.

Hypertrophy and hyperplasia of the rat pancreas produced by short-term dietary administration of soya-derived protein and soybean trypsin inhibitor.

Feeding soy protein concentrate to weanling rats over a one-week period produced a dose-related increase in pancreatic weight due to an increase in acinar cell size. Hyperplastic changes occur simultaneously, as evidenced by an increase in mitotic activity after two days on the test diet. Similar changes were also obtained by feeding soybean Kunitz trypsin inhibitor over the same time period. The results suggest that this approach may be useful as a model to investigate the effect of plant-derived material on the pancreas, in particular proliferative lesions.

Effects of dietary fats and soybean protein on azaserine-induced pancreatic carcinogenesis and plasma cholecystokinin in the rat.

Both dietary unsaturated fat and raw soybean products are known to enhance pancreatic carcinogenesis when fed during the postinitiation phase. A comparison of these two dietary components was made to evaluate the relative potency of each ingredient for enhancing pancreatic carcinogenesis and to determine if this enhancement was correlated with an increase in plasma cholecystokinin (CCK) levels. Male Wistar rats were initiated with a single dose of azaserine (30 mg/kg body weight) at 14 days of age. The rats were weaned to test diets formulated from purified ingredients. Dietary protein at 20% by weight was either casein or soy protein isolate (heat treated or raw). Corn oil was the unsaturated fat of major interest and it was fed at either 5 or 20% by weight. Pancreases were quantitatively evaluated for carcinogen-induced lesions at 2- and 4-month postinitiation. In a second experiment designed to closely mimic the above experiment, rats were implanted with cannulae which allowed plasma to be repetitively sampled over a 2.5-week period during which the test diets were fed. Plasma was collected both prior to introduction of the test diets and afterwards. Plasma CCK was measured by a specific radioimmunoassay. Both the 20% corn oil diet and the raw soy protein isolate diet enhanced pancreatic carcinogenesis. The effects of the raw soy protein isolate on the growth of the carcinogen-induced lesions were significantly greater than the effects of the 20% corn oil diet. Plasma CCK values were not elevated in the rats fed the 20% corn oil diet, but they were significantly elevated in the rats fed the raw soy protein isolate. Heat-treated soy protein isolate neither enhanced carcinogenesis nor elevated the plasma CCK level. This study demonstrates that certain plant proteins enhance the growth of carcinogen-induced pancreatic foci and that this effect is considerably greater than the enhancement by high levels of dietary unsaturated fat. Furthermore, the enhancement by the raw soy protein isolate may be mediated by CCK; but this does not appear to be the mechanism by which the unsaturated fat, corn oil, enhances pancreatic carcinogenesis.

Induction of ornithine decarboxylase activity in weanling rat pancreas by an orally administered soy protein isolate.

The induction of ornithine decarboxylase activity in weanling rat pancreas by a trypsin inhibitor-containing soy protein isolate has been studied. Oral administration of the isolate at 0.8, 1.6, 4.0, 6.0, and 8.0 mg/g body wt produced marked elevations in enzyme activity, a response which was proportional to the amount of isolate administered. Ornithine decarboxylase activity was measured at 2, 4, 6, 8, 12, and 24 hr after the isolate was given. A statistically significant increase in enzyme activity was evident as early as 2 hr after treatment; maximal activity occurred at 6 hr and was approximately 140 times greater than the

Liver and muscle proteolytic activity in male growing rats fed a field bean (Vicia faba L.) diet.

Total liver cathepsin A and D and gastrocnemius acid proteolytic activities have been evaluated in male growing rats fed ad libitum over periods of 15 and 30 days on 20%-protein diets containing either casein or raw field bean (Vicia faba L.) as protein sources. It has been found that, compared to the control casein-fed rats, those fed the legume diet exhibited a marked reduction (p less than 0.05) in the rate of growth and protein efficiency rate; liver proteolytic activity increased with ageing in the two dietary treatments and was found to be significantly higher (p less than 0.05) in the legume-fed rats. However, muscle proteolytic activity decreased with ageing and proved significantly increased (p less than 0.05) in the legume-fed rats.

Safety of trypsin inhibitors in the diet: effects on the rat pancreas of long-term feeding of soy flour and soy protein isolate.

The effects on the pancreas of chronic dietary exposure to defatted soy flour and soy protein isolate have been studied in two two-year feeding trials in rats. Emphasis was placed on detecting changes that might accompany low levels of dietary trypsin inhibitor (TI) as might be found in edible grade soy products and on studying the influence of protein nutrition. The major pathological findings in the pancreas were nodular hyperplasia (NH), consisting of foci of hyperplastic acinar cells often grossly visible by six months, and the benign neoplastic lesion, acinar adenoma (AA), which developed more slowly. In the first feeding trial, the objectives were to obtain the dose-response relationship of pancreatic pathology to dietary TI provided by raw and heated soy flour and to study the nutritional interaction of protein level which was varied from 10% to 30% using casein supplementation. Also, the responses to raw and heated soy protein isolate were compared to determine whether the removal of more than 50% of the constituents found in soy flour would alter the development of pancreatic lesions. In the second trial, the effect of unusually low levels of TI in raw and heat-treated soy protein isolate, prepared through a salt extraction process and fed at 10% and 30% protein in the diet, was investigated. The incidence of both NH and AA was positively related to the TI content of the diet. The probit transformation of the percent incidence of AA was linearly related to the log of TI/g protein in the diet. A single curve best described the response to 20% and 30% protein, with a slope that was distinctly greater than that for 10% protein. The intersection of the two curves near the TI concentration of edible grade soy flour predicts that protein level in the diet can be expected to have essentially no effect on the incidence of AA when TI activity is in this range. But, for proteins containing greater concentrations of TI, increasing the level of protein in the diet will increase the incidence of pancreatic pathology, while for proteins with quite low levels of TI, increasing the protein in the diet above 10% will have a protective effect.(ABSTRACT TRUNCATED AT 400 WORDS)

Enhancement of pancreatic carcinogenesis by raw soy protein isolate: quantitative rat model and nutritional considerations.

Foods containing soybean products have been shown to modify the biochemical and physiological status of the pancreas of several species of experimental animals. Recently, these products have been implicated as a factor in the causation of pancreatic neoplasms. Extensive experimental studies into the possible mechanisms need to be undertaken. Experimental details of a rat/azaserine model for the study of pancreatic carcinogenesis are reviewed. Emphasis is given to the quantitative components of this model and the adaptation of this model to the two-stage (initiation-promotion) concept of carcinogenesis. Particular attention is devoted to considerations of the experimental diets. Application of these concepts to the study of the postinitiational effects of raw and heated soybean protein isolate with and without the addition of high levels of unsaturated fat were undertaken. The results indicate that raw soybean isolate enhanced the growth of azaserine-induced pancreatic foci; whereas, a high level of unsaturated fat had a minimal effect. The effects of the soybean isolate were abolished by heat treatments, but the effects of the unsaturated fat would not be expected to be abolished by similar treatment with heat.

Antigenicity of native and modified Kunitz soybean trypsin inhibitors.

Food provides a continuous antigenic stimulus to the immune system and the antigenicity of processed food proteins should be considered in toxicological evaluations. The antigenicity of the Kunitz trypsin inhibitor was studied using antibodies prepared by inoculating rabbits with native, heat-denatured, and N-acetylcysteine-treated Kunitz soybean trypsin inhibitors. Immunochemical studies using a competitive solid-phase enzyme immunoassay and two groups of sera revealed two patterns of antigenicity. Antibodies elicited with the denatured inhibitor were specific for the denatured conformation of the protein. In contrast, native inhibitor elicited antibodies that selectively recognized determinants in both native and heat-treated protein, but that did not bind trypsin inhibitors treated with N-acetylcysteine. These results imply that: the disulfide bonds must be intact to maintain the native antigenic conformation and the cysteine treatment may suppress allergic manifestations of soybean trypsin inhibitors and possibly other food proteins. These studies were extended by analyzing a panel of monoclonal antibodies prepared against native Kunitz trypsin inhibitor. The inhibitor has at least two distinct antigenic sites (epitopes), one of which is retained under denaturing conditions. The measurement of native Kunitz trypsin inhibitor in food samples by immunoassay appears practical. The relevance of these findings to food processing, food safety, and health is also discussed.

The effects of long-term soy protein and milk protein feeding on the pancreas of Cebus albifrons monkeys.

Twenty-seven 2- to 4-yr-old cebus monkeys (Cebus albifrons) were fed from infancy purified diets containing lactalbumin, soy isolate, casein or soy concentrate as the sole protein source. Hematologic and clinical chemistry values were similar for all groups. Head and tail portions of each pancreas were surgically removed for histopathologic evaluation and determination of protein, RNA and DNA content, and for trypsin and chymotrypsin activity. Hematoxylin and eosin-stained sections from 26 of 27 monkeys showed normal pancreatic tissue with occasional acinar vacuolation in all diet groups. The remaining animal, one of only two fed soy concentrate, had diffuse interstitial fibrosis of the pancreas associated with mild to moderate atrophy of acinar tissue. Biochemical analyses of the pancreatic biopsies indicated no group differences among animals fed lactalbumin, soy isolate or casein. One of two monkeys in the soy concentrate group showed decreased pancreatic protein, RNA and trypsin concentrations; this was probably due to the fibrosis in this animal. No evidence of pancreatic hypertrophy or hyperplasia, as measured by RNA/DNA and protein/DNA ratios, respectively, was seen in any diet group.

Gallstone formation in hamsters: effect of varying animal and vegetable protein levels.

The lithogenic diet routinely used for production of gallstones in hamsters contains 20% casein. It has previously been shown that replacement of casein by soy protein significantly decreases gallstone formation. In this study hamsters were fed a lithogenic diet containing casein (C), soy isolate (S), C/S 3:1, C/S 1:1, and C/S 1:3. The percentages of hamsters with gallstones on these five diets were: 44, 12, 38, 23, and 15. Biliary cholesterol levels and lithogenic index both decreased significantly with increasing levels of soy protein. Dilution of casein with soy protein progressively decreases lithogenicity.

The lethal protein from Kintoki beans (Phaseolus vulgaris) identified as a lectin.

A glycoprotein isolated from Kintoki beans (Phaseolus vulgaris cultivar Kintoki) agglutinated human erythrocytes of all types and erythrocytes of rat, rabbit, sheep, and mouse. The lectin activity was not affected by 1 hr heating at 60 degrees C, but decreased slightly on heating for the same period at 70-80 degrees C and markedly at 90-100 degrees C. The activity was inhibited by galactose, lactose, N-acetyl galactosamine and fetuin. The inhibition was, however, weak, as often found for nonspecific lectins. The activity did not change when tyrosine residues or small parts of amino groups were modified, but decreased considerably when histidine residues or carboxyl groups were modified. This lectin was found to be relatively resistant to trypsin, and, particularly, to pepsin. All mice died within 48 hr when 200 microgram lectin per gram body weight was injected intraperitoneally and 14 microgram intravenously. The toxic activity changed in parallel with the lectin activity upon various treatments of the glycoprotein. In addition, blood analyses of injected mice suggested that the toxicity might be developed by the action of the lectin on blood cells.

Effects of alkali-treated proteins: feeding studies with free and protein-bound lysinoalanine in rats and other animals.

To find out whether alkali-treated proteins posses nephrotoxic properties, feeding studies were conducted with drastically treated soybean protein and casein, and also with lysinoalanine (LAL), the amino acid known to be formed in protein subjected to high pH at elevated temperature. The feeding of synthesized LAL to rats at dietary levels of 100 ppm and above induced typical renal changes, called nephrocytomegalia. No such changes or any other indications of toxicity were observed, however, upon feeding much higher levels of LAL (up to 6,000 ppm) when provided as the protein-bound compound in alkali-treated casein or soybean protein. When set free by complete acid hydrolysis, LAL induced considerable renal activity, comparable to that of the synthetic compound. These results indicate that alkali treatment of proteins does not induce nephrotoxic properties provided that the compound remains protein-bound. Some nephrotoxic activity was observed, however, with peptide-boound LAL in break-down products (molecular weight less than 5,000) of alkali-treated casein, but considerably less than that of the free compound. LAL-analyses in blood, urine, and feces of rats fed free or protein-bound LAL indicated a positive correlation between intestinal absorption and nephrotoxic potential. No renal changes were encountered upon feeding diets with 1,000 ppm synthetic LAL to mice, hamsters, rabbits, quail, dogs or monkeys, which suggest a species specificity of LAL-induced renal changes in rats.