Toxicological evaluation of morning glory seed: subchronic 90-day feeding study.

Diets containing 0.8, 2.53 and 8.0% field variety morning glory seed were fed to male and female rats (20 per group) in a 90-day subchronic feeding study. Gross clinical observations, body weight, and feed and water intake were recorded weekly. At 90 days, all surviving rats were autopsied, organs were weighed, and blood chemistry analyses, haematology, and bone-marrow evaluation for evidence of clastogenic effects were performed. Tissues from control (0% seed) and high-dose (8.0% seed) rats were examined histologically. Effects of morning glory seed were noted mainly in the high-dose group of both sexes. These included increases in mortality, feed consumption (on a body-weight basis), water consumption, serum alkaline phosphatase and potassium, white blood cell count, and brain and liver weights (as a percentage of body weight); body-weight gain and serum glucose were decreased. Significant changes seen in high-dose females alone were: increased haemoglobin, serum constituents (urea nitrogen, glutamic-pyruvic transaminase, glutamic-oxaloacetic transaminase, and ornithine carbamyl transferase), and organ weights (heart, kidney, spleen and pancreas as a percentage of body weight), and decreases in serum albumin, total protein, albumin:globulin ratio, and calcium. Significant changes occurring in high-dose males alone were: increased testicular weight (as a percentage of body weight), increased serum phosphorus, and decreased serum cholesterol. Liver degeneration in the high-dose females was greater than that in the controls. Mortality at 8.0% seed in the diet was 40% in males and 10% in females. At 0.8% seed, the only parameter that differed significantly from that of the controls was a final body-weight reduction in females without a corresponding reduction in feed consumption.

Toxicological evaluation of sicklepod and black nightshade seeds in short-term feeding studies in rats.

Nutritionally complete diets containing sicklepod or black nightshade seed at 1, 2, 4, 8, 16 and 32% were fed to groups of three to five male Sprague-Dawley rats in a series of short-term (8-9 days) toxicity studies. Gross clinical observations, body weights and feed and water intake data were recorded. Clinical chemistry analyses, haematology, histology and bone-marrow evaluation for evidence of clastogenic effects were performed. In addition, groups of five female rats were fed sicklepod seed at the same dosages to compare effects on body weight and feed and water consumption. For sicklepod, all of the animals fed diet containing 32% seed and one (female) fed diet containing 16% seed died by day 8. Body-weight gain and feed and water consumption were decreased with increasing doses of sicklepod seed. Other effects of sicklepod seed included: testicular hypospermia at dosages of 8% or greater, and bone-marrow depletion, reduced numbers of polychromatic erythrocytes in the bone marrow, increased neutrophil:lymphocyte ratio, and red nasal discharge at 16%. Black nightshade seed was relatively non-toxic compared with the sicklepod. The principal adverse effects of black nightshade were decreased body-weight gain and feed consumption, which occurred during the first 3 days of the study in animals fed 32% seed.

Toxicological and nutritional evaluation of velvetleaf seed: subchronic 90-day feeding study and protein efficiency ratio assay.

Velvetleaf seed, a common weed contaminant in grain, was fed to male and female rats (20 per group) in a 90-day subchronic feeding study. Diets contained 0, 2.5, 5.0 or 10.0% seed. Gross clinical observations, body weights, and feed and water intake data were recorded weekly. After 91-93 days, all of the animals were autopsied, organ weights were obtained, and clinical chemistry analyses, haematology and bone-marrow evaluation for evidence of clastogenic effects were performed. Tissues from control (0% seed) and high-dose animals were examined histologically. Few effects from velvetleaf seed in the diet were noted. Body-weight gain, water consumption, organ weights, bone marrow, and haematology measurements were similar to those of control rats. Male rats fed 2.5 or 10% seed consumed less feed/kg body weight than did the controls. For males fed 10% seed, the alkaline phosphatase concentration and albumin:globulin ratio were significantly increased compared with the 0% control values. For females fed 10% seed, serum glucose and cholesterol values were decreased compared with those for groups fed 0 or 2.5% seed. No histopathology was associated with ingestion of 10% velvetleaf seed. The protein quality (protein efficiency ratio) of velvetleaf seed, although lower than that of casein, was higher than values reported in the literature for corn, wheat and soya.

Pancreatic response in rats and mice to trypsin inhibitors from soy and potato after short- and long-term dietary exposure.

The effects on the pancreas of chronic (95 wk) dietary exposure to protease inhibitors from soy and potato were compared in rats and mice. Soy and potato trypsin inhibitor (TI) concentrates were prepared from defatted raw soy flour and potato juice, respectively, by selective precipitation and ultrafiltration. Animals were fed a diet in which casein supplied approximately 20% protein. Each concentrate (less than 1% of the diet) was added to provide 100 and 200 mg of trypsin inhibitor activity per 100 g of diet. In short-term (28 d) experiments in rats, both sources of TI decreased the apparent nutritional quality of casein and produced pancreatic hypertrophy consistent with a hormonally mediated feedback mechanism for pancreatic adaptation to diet that is interactive with the nutritional status of the animal. After long-term feeding (95 wk), soy and potato TI produced dose-related pancreatic pathology in rats consisting of nodular hyperplasia and acinar adenoma, which was typical of that associated with raw soy flour. Although mice responded similarly to rats to soy TI in short-term (28-d) feeding experiments, they were resistant to the formation of these lesions following long-term feeding. This considerable species variation in propensity to develop preneoplastic and neoplastic lesions of the pancreas is not predicted by the short-term hypertrophic and hyperplastic response of the pancreas to TI.

Toxicological evaluation of jimson weed (Datura stramonium) seed.

Diets containing 0.5, 1.58 and 5.0% jimson weed seed were fed to male and female rats (20/group) in a 90-day subchronic feeding study. The alkaloid content was 2.71 mg atropine and 0.66 mg scopolamine/g of seed. Gross clinical observations, body weights and feed and water intakes were recorded weekly. Tear production and pupil dilation measurements were made throughout the study. At 90 days, all of the animals were autopsied and clinical-chemistry analyses, complete haematology and bone-marrow evaluation for evidence of clastogenic effects were performed. Tissues from control (0% seed) and high-dose animals were examined histologically. The principal effects of jimson weed seed were: decreased body-weight gain, serum albumin and serum calcium; increased liver and testes weights (as a percentage of body weight), serum alkaline phosphatase and blood urea nitrogen. Female rats showed more marked responses to jimson weed seed than did males. In addition to the effects seen in both sexes, the females developed decreased serum total protein and cholesterol, and increased serum glutamic-pyruvic transaminase and chloride, red blood cell count, haemoglobin concentration and packed red cell volume. No histological lesions were associated with ingestion of jimson weed seed at 5.0%. It is concluded that jimson weed seed at concentrations of 0.5% or more in the diet produced adverse physiological changes in rats.

Nutritional value and safety of methionine derivatives, isomeric dipeptides and hydroxy analogs in mice.

Weight gains in mice fed amino acid diets containing methionine and 16 methionine derivatives and analogs were compared at graded dietary concentrations. Linear response was closely approximated for concentrations below those yielding maximum growth. Derivatization of L-methionine generally lowered potency, calculated as the ratio of the slopes of the two dose-response curves. However, the three isomeric dipeptides L-L-, L-D- and D-L-methionylmethionine, N-acetyl- and N-formyl-L-methionine, L-methionine sulfoxide and D-methionine were well utilized. The double derivative N-acetyl-L-methionine sulfoxide reduced potency below 60%. D-Methionine sulfoxide, N-acetyl-D-methionine and D-methionyl-D-methionine had potencies between 4 and 40%. The calcium salts of L- and D-alpha-hydroxy analogs of methionine had potencies of 55.4 and 85.7%, respectively. Several of the analogs were less growth-inhibiting or toxic at high concentrations in the diet than was L-methionine. These results imply that some methionine dipeptides or analogs may be better candidates for fortifying foods than L-methionine. Possible biochemical pathways for the utilization of methionine derivatives and analogs are also described.

Effect of sulfur amino acid supplementation of raw soy flour on the growth and pancreatic weights of rats.

Rat feeding studies showed that L-cystine, L-cysteine, N-acetyl-L-cysteine and L-methionine enhance the nutritional quality of raw (unheated) soy flour as measured in rats by the protein efficiency ratio (PER). Pancreas weights of rats fed diets supplemented with sulfur amino acids were higher than those of rats fed unsupplemented diets. In some instances this increase resulting from nutritional improvement of the diet was relatively greater than that in total body weight. Explanations are offered for these effects in terms of the proposed biofeedback mechanism involving the gastrointestinal endocrine system and the hormone cholecystokinin. The observed stimulation of pancreatic growth by added sulfur amino acids may be closely associated with this pancreatic regulatory mechanism when activated in response to dietary trypsin inhibitors present in unheated soy protein. The extent to which pancreatic hypertrophy develops as a result of a given exposure to trypsin inhibitors may be limited when the total amount or quality of the protein in the diet is less than optimal.

Nutritional value and safety in mice of proteins and their admixtures with carbohydrates and vitamin C after heating.

When a nutritionally complete basal diet containing 10% protein from casein was supplemented with 20% protein from unheated casein, wheat gluten or soy protein isolate, weanling mice exhibited significantly increased weight gains. In contrast, weight gains were markedly reduced compared to those with the basal diet; that is, growth was inhibited, when the supplement was soy protein or gluten that had been heated at 200 or 215 degrees C for 72 min in the dry state to stimulate crust baking. Addition of various carbohydrates to the gluten during heating prevented such growth inhibition. After heating with sodium ascorbate (but not L-ascorbic acid), soy protein (at 200 degrees C) and gluten (at 215 degrees C) completely prevented growth when added to the basal diet. Growth inhibition also occurred with a heated casein-ascorbate mixture, but was less than with the other proteins. The extent of growth inhibition increased sharply with temperature of heating in the range 180-215 degrees C and with sodium ascorbate concentration in the range 1-20%. Possible physical and chemical changes during heating of protein-ascorbate mixtures are discussed, as are possible mechanisms for the growth inhibition.

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)

Nutritional improvement of legume proteins through disulfide interchange.

Treatment of raw soy flour with L-cysteine or N-acetyl-L-cysteine results in the introduction of new half-cystine residues into sulfur-poor legume proteins, with a corresponding improvement in nutritional quality as measured by the protein efficiency ratio (PER) in rats. The proteins are modified through formation of mixed disulfide bonds among added sulfhydryl compounds, proteolytic enzyme inhibitors, and structural legume proteins. This modification leads to loss of inhibitory activity and increased protein digestibility and nutritive value. Sodium sulfite is more effective than cysteine in facilitating inactivation of trypsin inhibitors in soy flour. The synergistic effect of sodium sulfite and heat may be due to ability to induce rearrangement of protein disulfide bonds to produce new structural entities without altering the amino acid composition and to the fact that the new structures lose their ability to complex with trypsin or chymotrypsin. The same treatment inactivated hemagglutinins (lectins) in lima bean flour. These considerations suggest a key role for sulfur amino acids in the nutritional quality and safety of legumes.

Comparison of interlaboratory variation in amino acid analysis and rat growth assays for evaluating protein quality.

Estimates of inter- and intralaboratory variation of protein efficiency ratio (PER), relative PER (RPER), net protein ratio (NPR), relative NPR (RNPR), and nitrogen utilization (NU) were compared with those of amino acid analysis in the same batches of 7 protein sources (ANRC casein, egg white solids, minced beef, soy assay protein, rapeseed protein concentrate, pea flour, and whole wheat flour). Interlaboratory variation (estimated as between-laboratories coefficients of variation, CV) of NPR and RNPR (up to 6.0%) was lower than that of PER (up to 20.2%) and RPER (up to 18.5%). The interlaboratory determination of NPR and RNPR was also more reproducible than that of most essential amino acids (CV up to 10.0%), especially tryptophan (CV up to 23.7%), cystine (CV up to 17.6%), and methionine (CV up to 16.1%). Intralaboratory variation (estimated as within-laboratories CV) of amino acid analysis (up to 4.7%), however, was comparable to that of protein quality indices in most protein sources (up to 6.0%). The significant (P less than 0.01) positive correlations (r = 0.68-0.74) between amino acid scores and protein quality indices based on rat growth were further improved when amino acid scores were corrected for digestibility of protein (r = 0.73-0.78) or individual amino acids (r = 0.79-0.82).

The utilization and safety of isomeric sulfur-containing amino acids in mice.

Growth assays on a synthetic amino acid diet fed to mice showed that substituting D-methionine for the L-isomer resulted in a dose-dependent relative weight gain reaching approximately 76% when D-methionine was fed at a level equivalent to that optimal for the L-form. L-Cysteine and L-cystine stimulated growth in the presence of suboptimal levels of L-methionine, but D-cystine was growth depressing. L-Cystine is at least equally efficient in stimulating growth in the presence of D-methionine as in the presence of the L-isomer. Compared to a suboptimal (25% of maximum) level of L-methionine alone, supplements to this level L-methionine by N-acetyl-L-cysteine produced a weight gain of 214%; L-cysteine, 178%; L-cysteic acid, 154%; DL- + meso-lanthionine, 127%; L-cysteine sulfinic acid, 113%; D-cysteine, 76%; and S-methyl-L-cysteine, 13%. The observed growth-depressing effect of D-cysteine, D-cystine and S-methyl-L-cysteine at a concentration no greater than that optimal for methionine implies that these three sulfur-containing amino acids may be toxic. The results are discussed in terms of known and postulated transamination and transsulfuration pathways that govern the biological utilization of isomeric sulfur-containing amino acids and derivatives.

Nutritional improvement of soy flour.

Heating soy flour at 45 degrees C in pH 8.5 Tris buffer for 1 hour in the presence of cysteine or N-acetylcysteine followed by dialysis to remove unreacted thiols resulted in the introduction of new half-cystine residues and lowered trypsin inhibitor content from 37.5 to 6.8 or 4.8 mg/g, respectively, compared to only 14.8 mg/g in the absence of sulfhydryl compounds. Presence of cysteine or N-acetylcysteine during treatment increased protein efficiency ratio (PER) from 0.95 to 2.01 or 2.20, respectively. Corresponding values at 65 degrees C are 1.61, 2.43 and 2.02; and at 75 degrees C, 2.14, 2.53 and 3.19, respectively. Proteins are modified through formation of mixed disulfide bonds, which leads to loss of inhibitory activity and increased protein digestibility and nutritive value.

The nutritive value and safety of D-phenylalanine and D-tyrosine in mice.

Growth studies in mice fed synthetic amino acid diets showed that: 1) the biological utilization of D-phenylalanine relative to its L-isomer ranged from 28-81%, depending on the respective concentrations of these amino acids in the diet; 2) L-tyrosine can replace about one-half of the L-phenylalanine needed to achieve maximum weight gain and 3) D-tyrosine has no similar sparing effect on L-phenylalanine. In fact, addition of D-tyrosine to amino acid or casein diets depressed weight gain to 10% that of the control. This inhibition was significantly reduced by increasing the L-phenylalanine content of the amino acid diets and the protein content of the casein diets. Growth inhibition, therefore, appears to be related to the ratio of D-tyrosine to L-phenylalanine and possibly other amino acids in the diet, rather than to the absolute levels of D-tyrosine. Mechanistic rationalizations are offered to account for the toxic or antinutritional manifestation of D-tyrosine.

Inter- and intra-laboratory variability in rat growth assays for estimating protein quality of foods.

Protein efficiency ratio (PER), relative PER (RPER), net protein ratio (NPR), relative NPR (RNPR), and nitrogen utilization (NU) methods were investigated in an interlaboratory rat growth study. Six collaborating laboratories studied 7 protein sources (ANRC casein; minced beef; soya assay protein, SAP; pea flour; whole wheat flour, WW; rapeseed protein concentrate, RPC; and egg white solids, EW), and their 10 supplementary or complementary mixtures (casein + Met, SAP + Met, pea flour + Met, WW + Lys, WW + casein, WW + beef, WW + SAP, WW + pea flour, WW + RPC, WW + EW). Test protein(s) were added at the 8% level (N X 6.25). Casein + Met was used as the reference protein. Interlaboratory variation (estimated as between-laboratories coefficients of variation) of PER (up to 17.2%) was greater than that of RPER (up to 14.9%), NU (up to 9.5%), NPR (up to 7.0%), and RNPR, which had the lowest variability (up to 4.7%). In most cases, intralaboratory variation (estimated as within-laboratories coefficients of variation) for all the methods was less than 5%.

Composition and protein efficiency ratio of meat samples partially defatted with petroleum ether, acetone, or ethyl ether.

Freeze-dried beef samples were partially defatted with either petroleum ether, acetone, or ethyl ether before determination of protein efficiency ratio (PER) to study the extraction effects on the composition and protein nutritional quality of the extracted beef. Defatting a protein source, such as meat or a meat product, may often be necessary to produce a test diet that contains 10% protein and 8% fat. Amino acid, carnosine, anserine, creatine, creatinine, inosine, and proximate compositions were determined on the extracted samples. Resulting data were compared to the composition and PER data of the beef that had no solvent treatment. Although the chemical analysis data from the study showed some variation between the proteins and other nitrogenous components of the unextracted and the extracted beef, these variations were too small to affect the protein nutritional quality of the beef as measured by PER.

Protein-alkali reactions: chemistry, toxicology, and nutritional consequences.

Heat and alkali treatment of proteins catalyzes formation of crosslinked amino-acid side chains such as lysinoalanine, ornithino-alanine and lanthionine, and concurrent racemization of L-isomers of all amino acid residues to D-analogues. Factors that favor these transformations include high pH and temperature, long exposure, and certain inductive or steric properties of the various amino acid side chains. Factors that minimize crosslink formation include the presence of certain additives, such as cysteine or sulfite ions, and acylation of epsilon-NH2 groups of lysine side chains. Free and protein-bound lysinoalanine and D-serine induce nephrocytomegaly in rat kidney tissues. The presence of lysinoalanine and D-amino acid residues along a protein chain decreases its digestibility and nutritional quality. Understanding the factors that govern the formation of potentially harmful unnatural amino acid residues in food proteins and the toxic and nutritionally antagonistic action of these compounds in animals should lead to better and safer foods.

Bioavailability of some lysine derivatives in mice.

Growth assays using mice on synthetic amino acid diets showed that substituting epsilon-N-methyl-L-lysine, epsilon-N-dimethyl-L-lysine and epsilon-N-trimethyl-L-lysine for lysine resulted in relative replacement values about 1/12, 1/20 and 1/25, respectively, of that obtained with the standard lysine diet. Similar studies showed that the alpha-N-acetyl-L-lysine is not utilized by mice and that the relative the replacement value of epsilon-N-acetyl-L-lysine was about 3% of that of lysine. Analogous substitution of D-lysine and the lysine sulfur-containing analog, S-(2-aminoethyl)-L-cysteine, for lysine resulted in weight losses during the feeding period. The results are discussed with reference to factors that are expected to influence the biological utilization f lysine analogs and derivatives.