Background on international activities on protein quality assessment of foods.

The subject of protein quality assessment of foods and diets was addressed at the Codex Committee on Vegetable Proteins (1982-1989), FAO/WHO (1989, 2001) and WHO/FAO (2002) expert reviews. These international developments are summarized in this manuscript. In 1989, a Joint FAO/WHO Expert Consultation on Protein Quality Evaluation reviewed knowledge of protein quality assessment of foods, and specifically evaluated amino acid score corrected for protein digestibility, the method recommended by the Codex Committee on Vegetable Proteins. The report of the Consultation published in 1991 concluded that the Protein Digestibility-corrected Amino Acid Score (PDCAAS) method was the most suitable approach for routine evaluation of protein quality for humans. The Consultation recognized that the amino acid scoring pattern proposed by FAO/WHO/UNU (1985) for preschool children was at that time the most suitable pattern for calculating PDCAAS for all ages except infants in which case the amino acid composition of human milk was recommended to be the basis of the scoring pattern. The rat balance method was considered as the most suitable practical method for predicting protein digestibility by humans. Since its adoption by FAO/WHO (1991), the PDCAAS method has been criticised for a number of reasons. The FAO/WHO (2001) Working Group on analytical issues related to protein quality assessed the validity of criticisms of the PDCAAS method. While recognizing a distinct regulatory use of protein quality data, the Working Group recommended that the PDCAAS method may be inappropriate for the routine prediction of protein quality of novel and sole source foods which contain high levels of anti nutritional factors; and that for regulatory purposes, the method should be revised to permit values of >100 for high quality proteins. In evaluating the recommendations of the Working Group, the WHO/FAO (2002) Expert Consultation on Protein and Amino Acid Requirements endorsed the PDCAAS method with minor modifications to the calculation method but also raised several issues. These included the calculation of scoring patterns; prediction of amino acid digestibility by faecal and ileal methods; reduced bioavailability of lysine in processed proteins; truncation of the amino acid score and consequent PDCAAS value; protein digestibility as a first limiting factor in determining the overall available dietary nitrogen; and the calculation of amino acid score for a dietary protein mixture. These concerns were considered particularly important in relation to the regulatory aspects of protein quality of foods, and their resolution was urgently recommended through a new separate expert review.

Distribution of isoflavones in samples of serum, liver and mammary glands of rats or pigs fed dietary isoflavones.

BACKGROUND/AIMS: There has been great interest in the potential beneficial and adverse health effects of dietary isoflavones. Determination of tissue concentrations of isoflavone metabolites provides an insight into the potential bioactivity of dietary isoflavones. However, data on the distribution of isoflavones in animal models fed dietary isoflavones are limited. In this study, additional data on the distribution of isoflavones in serum and/or tissues of rats and pigs fed dietary isoflavones were generated. METHODS: Rats (male and female) were fed a casein control diet (containing no isoflavones) and an isoflavone-supplemented diet (containing an alcohol-washed soy protein isolate plus NOVASOY, providing a total of 1,047 mg/kg of total isoflavones). Female pigs were fed a control diet (without soy) containing 17.5 mg/kg of isoflavones, a soy diet containing 582.8 mg/kg of isoflavones or a soy diet supplemented with a daily dose of 2.3 g (equivalent to 42.0 and 14.5 mg/kg of body weight at the onset and end of treatment, respectively) of crystalline genistein. The concentrations of isoflavones in serum and tissues (liver and mammary gland) and in tissues (liver and mammary gland) of pigs were determined via a sensitive and rapid method using liquid chromatography/mass spectrometry. RESULTS: Rats fed the control diet containing no isoflavones had nondetectable levels of isoflavone metabolites in serum, liver and mammary gland samples. Rats fed the isoflavone-supplemented diet had the greatest levels of equol, followed by genistein, daidzein and glycitein, respectively, in their serum, livers and mammary glands. The concentrations of total isoflavones (daidzein, equol and genistein plus glycitein) in serum were significantly (p < 0.05) greater in male rats vs. female rats, but the reverse was true in the case of livers. Concentrations of daidzein, equol, genistein and glycitein were lowest (p < 0.05) in the livers of pigs fed the control diet, and in the mammary glands of female pigs there was only an effect of feeding soy plus genistein on the concentrations of daidzein and equol (p <0.05). CONCLUSIONS: The current data therefore show gender as well as species differences in the tissue distribution of isoflavones.

Sex- and age-specific immunomodulatory effects of dietary soya protein isolate and isoflavones in rats.

The present study examined, using rats as a model, the effects of sex and age of exposure to dietary soya components on serum total and soya-specific antibody content. In Expt 1, Sprague-Dawley rats at 50 d of age were fed diets containing 20 % casein or 20 % alcohol-washed soya protein isolate (SPI) with or without supplemental isoflavones (ISF, 250 mg/kg diet) for 70, 190 or 310 d. The offspring were fed the same diets as their parents. In Expt 2, juvenile Sprague-Dawley rats at 30 d of age were fed diets containing 20 % casein with or without supplemental ISF (50 mg/kg diet) or increasing amounts of alcohol-washed SPI (5, 10 or 20 %) for 90 d. Exposure of rats to dietary SPI before the age of 28 d increased serum total IgA and IgM, and induced the production of SPI-specific IgA, IgG, IgM and IgE antibodies. Feeding of juvenile or adult rats with SPI elevated serum total IgA in females, while the opposite occurred in males, and markedly stimulated the production of SPI-specific IgM in females and IgG in males. Our data suggest that the effects of soya proteins and ISF on the production of serum total and SPI-specific antibodies appear to be sex dependent and also related to the age of exposure to soya in rats. However, the physiological significance of these immune responses remains to be determined.

Amino acid analysis.

Amino acid analysis is used to determine the amino acid content of amino acid-, peptide- and protein-containing samples. With minor exceptions, proteins are long linear polymers of amino acids connected to each other via peptide bonds. The first step of amino acid analysis involves hydrolyzing these peptide bonds. The liberated amino acids are then separated, detected, and quantified. The method was first developed by Moore, Stein and coworkers in the 1950s using HCl acid hydrolysis, and, despite considerable effort by many workers, the basic methodology remains relatively unchanged. This unit provides an overview and strategic planning for amino acid analysis, discussing a range of methodologies and issues. In addition, several common methods used for analysis of L-amino acids are described in detail, including: HCl acid hydrolysis, performic acid oxidation for methionine and cysteine analysis, base hydrolysis for tryptophan analysis, analysis of free amino acids, and analysis of reactive lysine.

Effects of source of protein and supplementary extracted isoflavones and anthocyanins on longevity of Stroke-prone Spontaneously Hypertensive (SHRSP) rats.

Amount of dietary protein is known to influence blood pressure in humans and animal models. However, contradictory reports are available on the influence of source of dietary protein and soy isoflavones on blood pressure. Information on potential effect of anthocyanins, potent flavonoid antioxidants widely distributed in fruits and vegetables, on hypertension is also limited. Therefore, this study was conducted to examine whether source of dietary protein (casein vs. soybean protein, washed by alcohol to remove most isoflavones), dietary extracted isoflavones and anthocyanins modulate the lifespan of Stroke-prone Spontaneously Hypertensive (SHRSP) rats, one of the most suitable models for hemorrhagic stroke. Body weight and systolic blood-pressure matched groups of 47 day-old SHRSP rats (n = 16) received semi-purified diets containing 200 g/kg protein (casein or soybean) supplemented with 0 or 500 mg/kg isoflavones (NOVASOY, a commercial soy isoflavones supplement extracted from soybean), and 0 or 500 mg/kg anthocyanins (extracted from elderberry). The drinking water contained 10 g/l sodium chloride to induce early hypertension. Survival times and survival rates of rats were determined. The survival rates were determined for each group and expressed as a percentage of the original number of rats still alive on a given day. The survival times and survival rates of animals fed casein and soybean protein diets were not different (P > 0.05). However, there was a significant effect of supplementation with isoflavones or anthocyanins on survival times and survival rates. Death occurred significantly earlier (P < 0.05) in the isoflavones- or anthocyanins-supplemented groups.

Effect of glycosidation of isoflavones on their bioavailability and pharmacokinetics in aged male rats.

There are limited reports on the bioavailability and pharmacokinetics of isoflavones in elderly humans and aged animals. The present study was conducted to assess the effect of glycosidation of isoflavones on their bioavailability and pharmacokinetics in aged (20 month old) male Fischer-344 (F-344) rats. The F-344 rat, developed by the National Institute on Aging, is an inbred rat model that is commonly used for aging studies and resembles many features of aging humans. Three sources of isoflavones; Novasoy (a commercial supplement), a mixture of synthetic aglycons (daidzein, genistein and glycitein), and a mixture of synthetic glucosides (daidzin, genistin, and glycitin) were tested. Following administration, blood samples were collected at different times (0-48 h post-oral gavage and 0-8 h post-IV dosing). Plasma isoflavones and 7-hydroxy-3-(4'-hydroxyphenyl)-chroman (a metabolite of daidzein) were measured by LC/MS. The extent of absorption was determined by comparing the area under the curve (AUC) of the plasma-concentration time curve after intravenous (IV) administration with that following oral administration. The extent of bioavailability was then calculated as: %bioabailability = (AUC(or)/AUC(IV))x(Dose(IV)/Dose(or))x100. Bioavailabilities for genistein were significantly (p = 0.013) higher for the aglycon (35 +/- 9%) compared with the glucoside forms (11 +/- 3%). In contrast, the bioavailabilities for glycitein were significantly (p = 0.011) higher in Novasoy (27 +/- 13%) and the glucoside form (21 +/- 10%) compared with the aglycon (8 +/- 3%). No significant differences in the bioavailability of daidzein were observed in aged rats dosed with aglycon, glucoside or Novasoy. However, aged rats were able to produce equol as early as 8 h post-dosing. In summary, the source of isoflavones had significant effects on genistein and glycitein bioavailability in aged male rats.

Need for accurate and standardized determination of amino acids and bioactive peptides for evaluating protein quality and potential health effects of foods and dietary supplements.

Accurate standardized methods for the determination of amino acid in foods are required to assess the nutritional safety and compositional adequacy of sole source foods such as infant formulas and enteral nutritionals, and protein and amino acid supplements and their hydrolysates, and to assess protein claims of foods. Protein digestibility-corrected amino acid score (PDCAAS), which requires information on amino acid composition, is the official method for assessing protein claims of foods and supplements sold in the United States. PDCAAS has also been adopted internationally as the most suitable method for routine evaluation of protein quality of foods by the Food and Agriculture Organization/World Health Organization. Standardized methods for analysis of amino acids by ion-exchange chromatography have been developed. However, there is a need to develop validated methods of amino acid analysis in foods using liquid chromatographic techniques, which have replaced ion-exchange methods for quantifying amino acids in most laboratories. Bioactive peptides from animal and plant proteins have been found to potentially impact human health. A wide range of physiological effects, including blood pressure-lowering effects, cholesterol-lowering ability, antithrombotic effects, enhancement of mineral absorption, and immunomodulatory effects have been described for bioactive peptides. There is considerable commercial interest in developing functional foods containing bioactive peptides. There is also a need to develop accurate standardized methods for the characterization (amino acid sequencing) and quantification of bioactive peptides and to carry out dose-response studies in animal models and clinical trials to assess safety, potential allergenicity, potential intolerance, and efficacy of bioactive peptides. Information from these studies is needed for determining the upper safe levels of bioactive peptides and as the basis for developing potential health claims for bioactive peptides. This information is, in turn, needed by regulatory agencies for developing appropriate policy and regulations on adding these substances to foods and for determining if health claims are scientifically substantiated.

Impact of foods nutritionally enhanced through biotechnology in alleviating malnutrition in developing countries.

According to United Nations (UN) projections, the world's population will grow from 6.1 billion in 2000 to 8 billion in 2025 and 9.4 billion in 2050. Most (93%) of the increase will take place in developing countries. The rapid population growth in developing countries creates major challenges for governments regarding food and nutrition security. According to current World Health Organization estimates, more than 3 billion people worldwide, especially in developing countries, are malnourished in essential nutrients. Malnutrition imposes severe costs on a country's population due to impaired physical and cognitive abilities and reduced ability to work. Little progress has been made in improving malnutrition over the past few decades. The Food and Agriculture Organization of the UN would like to see more nutrient-rich foods introduced into these countries, because supplements are expensive and difficult to distribute widely. Biofortification of staple crops through modern biotechnology can potentially help in alleviating malnutrition in developing countries. Several genetically modified crops, including rice, potatoes, oilseeds, and cassava, with elevated levels of essential nutrients (such as vitamin A, iron, zinc, protein and essential amino acids, and essential fatty acids); reduced levels of antinutritional factors (such as cyanogens, phytates, and glycoalkaloid); and increased levels of factors that influence bioavailability and utilization of essential nutrients (such as cysteine residues) are advancing through field trial stage and regulatory processes towards commercialization. The ready availability and consumption of the biofortified crops would have a significant impact in reducing malnutrition and the risk of chronic disease in developing countries.

Consumption of soy protein isolate modulates the phosphorylation status of hepatic ATPase/ATP synthase beta protein and increases ATPase activity in rats.

ATPase/ATP synthase plays important roles in the regulation of carbohydrate, protein, and lipid metabolism through modulating energy homeostasis. The purpose of this study was to examine the effects of feeding soy proteins and isoflavones (ISF) on the enzymatic activity and protein modification of hepatic mitochondrial ATPase/ATP synthase. In Expt. 1, Sprague-Dawley rats aged 50 d were fed diets containing either 20% casein or 20% alcohol-washed soy protein isolate (SPI) with or without supplemental ISF (770.7 micromol/kg diet) for 70 d. In Expt. 2, weanling Sprague-Dawley rats were fed diets containing 20% casein with or without added ISF (154.1 micromol/kg diet) or 20% SPI for 90 d. Hepatic mitochondrial ATPase activity was significantly higher in the rats fed SPI than in those fed casein. Addition of ISF to SPI eliminated the action of SPI. ATPase/ATP synthase beta protein contents in the liver were unchanged; however, its patterns measured by 2-dimensional Western blot were different among dietary groups. The rats fed SPI or SPI plus ISF had 3 more major protein spots with the same molecular weights (80 kDa and 55 kDa) as those presented in the rats fed casein but with different isoelectric points. Pretreatment of hepatic mitochondrial proteins from the rats fed casein with alkaline phosphatase produced the same ATPase/ATP synthase beta patterns as observed in the SPI-fed rats and significantly elevated the ATPase activity. These results suggest that consumption of soy proteins increases hepatic ATPase activity, which might be a consequence of increased dephosphorylation or decreased phosphorylation of the mitochondrial ATPase/ATP synthase beta protein.

Bioavailability of soy isoflavones in rats Part I: application of accurate methodology for studying the effects of gender and source of isoflavones.

There are limited and controversial reports about the effects of gender and source of isoflavones on their bioavailability. Moreover, several previous studies have not used appropriate methodology to determine the bioavailability of soy isoflavones, which requires comparing the area under the plasma concentration-time curve after both oral and intravenous injection (IV) administration. Therefore, the present study was conducted to determine the bioavailability of isoflavones from different sources following both oral and IV administration in male and female rats. Three sources of isoflavones; Novasoy (a commercial supplement), a mixture of synthetic aglycones (daidzein, genistein and glycitein) and a mixture of synthetic glucosides (daidzin, genistin and glycitin) were tested. Following administration, blood samples were collected at several time points (0, 10, 30 min and 1, 2, 8, 24, 48 h post oral gavage and 0, 10, 30, 45 min and 1, 2, 3, 4, 8 h post-IV dosing) and plasma isoflavones were measured by LC/MS. Bioavailability values for daidzein, genistein and glycitein were significantly (p <0.05) higher (up to sevenfold) in Novasoy and the glucoside forms of isoflavones compared with those of the aglycone forms. Moreover, significant (p <0.05) gender differences in the bioavailability of 7-hydroxyl-3-(4'-hydroxyphenyl)-chroman (a metabolite of daidzein), glycitein and daidzein were observed for Novasoy, with higher values in male rats. In summary, the source of isoflavones and the sex of rats had significant effects on isoflavone bioavailability.

Dietary soy protein isolate modifies hepatic retinoic acid receptor-beta proteins and inhibits their DNA binding activity in rats.

Retinoic acid receptors (RAR) belong to the same nuclear receptor superfamily as thyroid hormone receptors (TR) that were previously shown to be modulated by dietary soy protein isolate (SPI). This study has examined the effect of dietary SPI and isoflavones (ISF) on hepatic RAR gene expression and DNA binding activity. In Expt. 1, Sprague-Dawley rats were fed diets containing 20% casein or 20% alcohol-washed SPI in the absence or presence of increasing amounts of ISF (5-1250 mg/kg diet) for 70, 190, or 310 d. In Expt. 2, weanling Sprague-Dawley rats were fed diets containing 20% casein with or without supplemental ISF (50 mg/kg diet) or increasing amounts of alcohol-washed SPI (5, 10, and 20%) for 90 d. Intake of soy proteins significantly elevated hepatic RARbeta2 protein content dose-dependently compared with a casein diet, whereas supplemental ISF had no consistent effect. Neither RARbeta protein in the other tissues measured nor the other RAR (RARalpha and RARgamma) in the liver were affected by dietary SPI, indicating a tissue and isoform-specific effect of SPI. RARbeta2 mRNA abundances were not different between dietary groups except that its expression was markedly suppressed in male rats fed SPI for 310 d. DNA binding activity of nuclear RARbeta was significantly attenuated and the isoelectric points of RARbeta2 were shifted by dietary SPI. Overall, these results show for the first time, to our knowledge, that dietary soy proteins affect hepatic RARbeta2 protein content and RARbeta DNA binding activity, which may contribute to the suppression of retinoid-induced hypertriglyceridemia by SPI as reported.

An accurate and reproducible method for the quantitative analysis of isoflavones and their metabolites in rat plasma using liquid chromatography/mass spectrometry combined with photodiode array detection.

To study the safety and potential health benefits of soy isoflavones, a rapid and simple method based on liquid chromatography combined with mass spectrometry (LC/MS) and photodiode array detector (PDA) was developed for the determination of isoflavones in rat plasma. The analytes included daidzein, genistein, glycitein, equol, 4-ethyl phenol, and biochanin A over a concentration range of 1.0-4320.0 nM using 75 microL of rat plasma. Rat plasma samples were hydrolyzed by adding an enzyme mixture from Helix pomatia containing glucuronidase and sulfatase to convert the isoflavone beta-glycosides daidzin, genistin, and glycitin to their active aglycone forms. A liquid-liquid extraction method using ethyl acetate as the extraction solvent was used to extract aglycones and the internal standards (phenolphthalein beta-D glucuronide, 4-methylumbelliferyl sulfate, and apigenin) from digested plasma samples. The extract was evaporated to dryness under a nitrogen stream, reconstituted with 0.1% formic acid in water-acetonitrile (85 + 15), and injected into a Zorbax SB-CN reversed-phase column (4.6 x 75 mm, 3.5 microm particle size). The Micromass ZQ detector was operated in the positive ion selected-ion monitoring mode. The flow rate for LC was 1.0 mL/min, with a split where 25% of the effluent was introduced into the electrospray ionization probe of the MS instrument and 75% into the PDA. The chromatographic run time was 16.0 min, with delay of 10 min/injection. The interday precision and accuracy of the standard samples were <2.6% relative standard deviation and <10% relative error, respectively. Recovery of the reported isoflavones with this method varied from 86 to 100%.

Nuclear receptors: potential biomarkers for assessing physiological functions of soy proteins and phytoestrogens.

Soy consumption is associated with decreased incidence of chronic diseases, including cardiovascular diseases, atherosclerosis, diabetes, osteoporosis, and certain types of cancers. However, consumption of high amounts of soy isoflavones may adversely influence endocrine functions, such as thyroid function and reproductive performance, because of their structural similarity to endogenous estrogens. Nuclear receptors are a group of transcription factors that play critical roles in the regulation of gene expression and physiological functions through direct interaction with target genes. Modulation of the abundance of these receptors, such as changing their gene expression, alters the sensitivity of the target cells or tissues to the stimulation of ligands, and eventually affects the relevant physiological functions, such as growth, development, osteogenesis, immune response, lipogenesis, reproductive process, and anticarcinogenesis. A number of studies have shown that the bioactive components in soy can modify the expression of these receptors in various tissues and cancer cells, which is believed to be a key intracellular mechanism by which soy components affect physiological functions. This review summarizes the current understanding of the modulation of nuclear receptors by soy proteins and isoflavones, and focuses especially on the receptors for estrogens, progesterone, androgen, vitamin D, retinoic acid, and thyroid hormones as well as the potential impact on physiological functions.

Tissue-specific regulation of acetyl-CoA carboxylase gene expression by dietary soya protein isolate in rats.

We have recently reported that intake of soya protein isolate (SPI) inhibited the DNA-binding activities of hepatic thyroid hormone receptor (TR). The genes for acetyl-CoA carboxylase (ACC), a rate-limiting enzyme in fatty acid synthesis, contain the thyroid hormone response element in their promoters and are regulated by TR. The present study has examined the effect of long-term feeding of SPI and soya isoflavones (ISF) on the gene expression and protein phosphorylation of different ACC isoforms in different tissues and plasma triacylglycerol (TAG) levels in rats. Sprague-Dawley female rats were fed diets containing 20 % casein or alcohol-washed SPI with or without supplemental ISF for 70, 190 and 310 d. SPI intake significantly reduced plasma TAG concentrations compared with casein, whereas supplemental ISF had no effect. Hepatic ACCalpha and ACCbeta mRNA abundance and protein content were markedly lower in the rats fed SPI than in those fed casein. The protein contents of ACCalpha in the kidney and ACCbeta, the predominant isoform in the heart and kidney, were unchanged by dietary SPI. The ratios of phospho-ACCalpha/ACCalpha and phospho-ACCbeta/ACCbeta were not different among dietary groups in all tissues measured. The present study demonstrates that ingestion of SPI decreases plasma TAG level and down-regulates ACCalpha and ACCbeta gene expression in the liver but not in the heart and kidney. The results indicate that the effect of SPI is tissue-specific and that alteration of ACC gene expression rather than phosphorylation status may play a major role in the regulation of ACC activities by soya proteins.

Chromatographic determination of amino acids in foods.

Amino acids in foods exist in a free form or bound in peptides, proteins, or nonpeptide bonded polymers. Naturally occurring L-amino acids are required for protein synthesis and are precursors for essential molecules, such as co-enzymes and nucleic acids. Nonprotein amino acids may also occur in animal tissues as metabolic intermediates or have other important functions. The development of bacterially derived food proteins, genetically modified foods, and new methods of food processing; the production of amino acids for food fortification; and the introduction of new plant food sources have meant that protein amino acids and amino acid enantiomers in foods can have both nutritional and safety implications for humans. There is, therefore, a need for the rapid and accurate determination of amino acids in foods. Determination of the total amino acid content of foods requires protein hydrolysis by various means that must take into account variations in stability of individual amino acids and resistance of different peptide bonds to the hydrolysis procedures. Modern methods for separation and quantitation of free amino acids either before or after protein hydrolysis include ion exchange chromatography, high performance liquid chromatography (LC), gas chromatography, and capillary electrophoresis. Chemical derivatization of amino acids may be required to change them into forms amenable to separation by the various chromatographic methods or to create derivatives with properties, such as fluorescence, that improve their detection. Official methods for hydrolysis and analysis of amino acids in foods for nutritional purposes have been established. LC is currently the most widely used analytical technique, although there is a need for collaborative testing of methods available. Newer developments in chromatographic methodology and detector technology have reduced sample and reagent requirements and improved identification, resolution, and sensitivity of amino acid analyses of food samples.

Effects of antinutritional factors on protein digestibility and amino acid availability in foods.

Digestibility of protein in traditional diets from developing countries such as India, Guatemala, and Brazil is considerably lower compared to that of protein in typical North American diets (54-78 versus 88-94%). The presence of less digestible protein fractions, high levels of insoluble fiber, and high concentrations of antinutritional factors in the diets of developing countries, which are based on less refined cereals and grain legumes as major sources of protein, are responsible for poor digestibility of protein. The effects of the presence of some of the important antinutritional factors on protein and amino digestibilities of food and feed products are reviewed in this chapter. Food and feed products may contain a number of antinutritional factors that may adversely affect protein digestibility and amino acid availability. Antinutritional factors may occur naturally, such as glucosinolates in mustard and rapeseed protein products, trypsin inhibitors and hemagglutinins in legumes, tannins in legumes and cereals, phytates in cereals and oilseeds, and gossypol in cottonseed protein products. Antinutritional factors may also be formed during heat/alkaline processing of protein products, yielding Maillard compounds, oxidized forms of sulfur amino acids, D-amino acids, and lysinoalanine (LAL, an unnatural amino acid derivative). The presence of high levels of dietary trypsin inhibitors from soybeans, kidney beans, or other grain legumes can cause substantial reductions in protein and amino acid digestibilities (up to 50%) in rats and pigs. Similarly, the presence of high levels of tannins in cereals, such as sorghum, and grain legumes, such as fababean (Vicia faba L.), can result in significantly reduced protein and amino acid digestibilities (up to 23%) in rats, poultry, and pigs. Studies involving phytase supplementation of production rations for swine or poultry have provided indirect evidence that normally encountered levels of phytates in cereals and legumes can reduce protein and amino acid digestibilities by up to 10%. D-amino acids and LAL formed during alkaline/heat treatment of proteins such as casein, lactalbumin, soy protein isolate, or wheat proteins are poorly digestible (less than 40%), and their presence can reduce protein digestibility by up to 28% in rats and pigs. A comparison of the protein digestibility determination in young (5-week) versus old (20-month) rats suggests greater susceptibility to the adverse effects of antinutritional factors in old rats than in young rats. Therefore, the inclusion of protein digestibility data obtained with young rats, as the recommended animal model, in the calculation of PDCAAS (Protein Digestibility-Corrected Amino Acid Score) may overestimate protein digestibility and quality of products, especially those containing antinutritional factors, for the elderly. For products specifically intended for the elderly, protein digestibility should be determined using more mature rats.

Soy protein isolate increases hepatic thyroid hormone receptor content and inhibits its binding to target genes in rats.

Our previous studies showed that intake of 20% alcohol-washed soy protein isolate (SPI) significantly increased hepatic thyroid hormone receptor (TR) beta1 protein content in rats. However, whether SPI influences the binding ability of TR to its target genes is unknown. The purpose of this study was to examine the effect of increasing amounts of dietary SPI on hepatic TRbeta1 content and the binding of TR to thyroid hormone response element (TRE) in rats. Sprague-Dawley rats (28 d old) were fed diets containing casein (20%) with or without isoflavone supplementation (50 mg/kg diet) or alcohol-washed SPI (5, 10, or 20%) for 90 d. The hepatic TRbeta1 protein content was measured by Western blot, and the binding ability of TR to DNA was examined by electrophoretic mobility shift assay. Consumption of the 20% SPI diet increased pancreatic relative weight and decreased spleen relative weight. Intake of SPI markedly elevated TRbeta1 content in both male and female rats compared with a casein-based control diet. The increase in TRbeta1 in females was much higher than that in males. Interestingly, the binding abilities of TR to DNA were significantly inhibited by increasing amounts of dietary SPI in female rats. In conclusion, this study shows for the first time that dietary SPI increases hepatic TRbeta1 protein content and inhibits the binding of TR to target genes. Modulation of hepatic TRbeta1, a key regulator of gene expression involved in lipid metabolism, by SPI may be a novel mechanism by which soy components lower blood lipid level and exert their hypocholesterolemic actions.