Low dietary carbohydrate induces structural alterations in enterocytes of the chicken ileum.

We investigated the role of dietary carbohydrates in the maintenance of the enterocyte microvillar structure in the chicken ileum. Male chickens were divided into the control and three experimental groups, and the experimental groups were fed diets containing 50%, 25%, and 0% carbohydrates of the control diet. The structural alterations in enterocytes were examined using transmission electron microscopy and immunofluorescent techniques for ß-actin and villin. Glucagon-like peptide (GLP)-2 and proglucagon mRNA were detected by immunohistochemistry and in situ hybridization, respectively. Fragmentation and wide gap spaces were frequently observed in the microvilli of the 25% and 0% groups. The length, width, and density of microvilli were also decreased in the experimental groups. The experimental groups had shorter terminal web extensions, and there were substantial changes in the mitochondrial density between the control and experimental groups. Intensities of ß-actin and villin immunofluorescence observed on the apical surface of enterocytes were lower in the 0% group. The frequency of GLP-2-immunoreactive and proglucagon mRNA-expressing cells decreased with declining dietary carbohydrate levels. This study revealed that dietary carbohydrates contribute to the structural maintenance of enterocyte microvilli in the chicken ileum. The data from immunohistochemistry and in situ hybridization assays suggest the participation of GLP-2 in this maintenance system.

Dietary carbohydrate modifies the density of L cells in the chicken ileum.

Glucagon-like peptides (GLPs) are secreted from intestinal L cells and stimulate various physiological functions in the gastrointestinal tract. The secretion of GLPs is influenced by macronutrient ingestion. This study aims to clarify the effects of dietary carbohydrate (CHO) on L cells in the chicken ileum. Six-week-old, male White Leghorn chickens were divided into three groups: control, low-CHO and CHO-free, with five chickens in each group. Paraffin sections were made from the proximal and distal ileum of each animal and subjected to immunohistochemistry for GLP-1 and GLP-2 peptides and in situ hybridization for proglucagon (PG) mRNA. A significant reduction of GLP-1- and GLP-2-immunoreactive cells was observed in the two experimental groups compared with that in the control. A reduction of cells expressing PG mRNA was observed in the proximal and distal ileum of the CHO-free group compared with that in the control. The ratio of GLP-1-immunoreactive cells showing Ki-67 immunoreactivity was significantly lower in the distal ileum of the CHO-free group than that in the control group. These data suggest that dietary CHO is an effective stimulator for modifying L cell density in the chicken ileum.

Nutrient digestibility, fecal output of fractionated proteins, and ruminal fermentation parameters of goats fed a diet supplemented with spent green tea and black tea leaf silage.

Spent green and black tea leaf silage (GTS and BTS, respectively) was offered as a protein supplement to goats to examine in vivo digestibility, nitrogen balance, urinary excretion of purine derivatives, and ruminal fermentation. Four castrated goats were fed a basal diet supplemented with alfalfa hay cube (AHC), GTS, or BTS in a 4 × 4 Latin square design. Digestibilities of various nutrients except for nitrogen (N) fraction were unaffected by the type of supplement. Digestibility of acid detergent insoluble N (ADIN) in BTS treatment was a negative value and significantly lower than those in other treatments. Urinary N output and retained N were not significantly affected by the diets. The fecal output of neutral detergent insoluble nitrogen (NDIN) and ADIN in the BTS treatment was significantly higher than those in other treatments. Urinary excretion of purine derivatives was not affected by the treatments. Ruminal NH3 -N concentration in AHC and GTS treatments were not significantly different, but that in the BTS treatment was significantly lower than others. These results indicated that GTS is substitutable for AHC as a protein supplement, whereas BTS was able to bind proteins tightly in the digestive tract, which lowered ruminal N degradability and increase fecal N output.

Glycated Tryptophan PHP-THßC Incorporated into Various Chicken Embryonic Cells Constitutes Cellular Proteins.

Glycation is a non-enzymatic reaction, and amino acids are glycated by glucose in vivo. Tryptophan is glycated with glucose to form two types of glycated compounds, tryptophan-Amadori product and (1R, 3S)-1-(D-gluco-1, 2, 3, 4, 5-pentahydroxypentyl)-1, 2, 3, 4-tetrahydro-ß-carboline-3-carboxylic acid (PHP-THßC). Although PHP-THßC can be incorporated into various chicken embryonic cells, the mechanism of its incorporation into intracellular fluids has not been clarified. In this study, we examined whether PHP-THßC once incorporated into various chicken embryonic cells can combine with proteins. Embryonic cells from the breast muscle, liver, spleen, kidney, proventriculus, gizzard, and skin were prepared and 3H-PHP-THßC was added to the culture medium at final concentrations of 0, 200, 400, 600, and 800 µM to examine the incorporation of PHP-THßC. After 18 h of incubation, radioactivity was measured in the whole-cell and protein fractions of the chicken embryonic cells. As PHP-THßC concentration increased from 0 to 600 µM, its accumulation in the whole-cell fractions of all types of chicken embryonic cells linearly increased and reached the maximum level. The saturated PHP-THßC accumulation in the whole-cell fractions suggests that PHP-THßC could be incorporated into intracellular fluids across cellular membranes by some transporter proteins. As PHP-THßC concentration increased from 0 to 800 µM, its accumulation in the protein fractions of all types of chicken embryonic cells increased in a linear manner and reached a maximum level in the 800 µM PHPTHßC treatment group. This is the first study to indicate that a part of PHP-THßC incorporated into the whole-cell fraction was detected in the protein fraction of various chicken embryonic cells.

Influence of Dietary Metformin on the Growth Performance and Plasma Concentrations of Amino Acids and Advanced Glycation End Products in Two Types of Chickens.

Glycation is a non-enzymatic reaction inducing the bonding of glucose to amino acids and proteins. Glycated amino acids are not useful for protein synthesis, suggesting that glycation reduces the utilization of amino acids. Metformin (MF) is well known as a therapeutic drug for type II diabetes that inhibits glycation. It is possible that treatment with MF raises the utilization of amino acids by the inhibition of glycation, thereby improving the growth performance of chickens. In the present study, therefore, we investigated the influence of dietary MF on the growth performance, and plasma concentrations of free amino acids and N ε -(Carboxymethyl)lysine (CML), which is an advanced glycation end product, in layer (Experiment 1) and broiler (Experiment 2) chickens. From 7 d of age, chicks were allowed free access to one of the experimental diets containing MF at 3 supplementation levels (0, 150, and 300 mg/kg diet) for 14 days. Body weight and feed intake were measured every week. At the end of the experiments, blood and breast muscle (M. pectoralis major) were collected for further analysis. Dietary MF did not affect weight gain, feed intake, or feed efficiency in both layer and broiler chickens. Dietary MF at the level of 150 mg/kg diet increased breast muscle weight in both layer and broiler chickens. Dietary MF increased plasma concentrations of branched chain amino acids and decreased concentrations of CML in layer chickens, although it did not affect plasma concentrations of glucose. The present study suggested that dietary MF might have the potency to increase breast muscle weight of layer chickens with an increment in plasma concentrations of branched-chain amino acids.

Dietary carbohydrate effects on histological features of ileal mucosa in White Leghorn chicken.

White Leghorn chickens were divided into the control, low-carbohydrate (CHO), and CHO-free groups to investigate dietary CHO's significance on histological features of chicken ileal mucosa. Paraffin sections of distal ileum from each chicken were stained by periodic acid-Schiff reaction and subjected to morphometrical analysis. Most villi in the control group had a fingerlike shape but those of the experimental groups showed irregular shapes. Villus height, crypt depth and the number of mitotic cells per crypt were significantly lower in the CHO-free group than in the control group. The density of goblet cells also showed a significant decreasing trend with a reduction in dietary CHO level. In conclusion, dietary CHO positively affects the proliferation of epithelial cells in the chicken ileum.

Dietary Protein Level Influences on Neurotensin-immunoreactive Cells in the Chicken Ileum.

Neurotensin is secreted from intestinal N cells in response to the food ingestion. Influences of different dietary protein levels on neurotensin-immunoreactive cells in the chicken ileum were examined by using immunohistochemical and morphometrical techniques. The results showed that dietary protein had an obvious influence on neurotensin-immunoreactive cells in the chicken ileum. Four experimental groups were used, with dietary crude protein (CP) levels of 18% (control), 9%, 4.5% and 0%. Enteroendocrine cells showing neurotensin-immunoreactivity were located in crypts and villous epithelium in all groups. Most of the neurotensin-immunoreactive cells in the villous epithelium showed pyramidal or spindle-like shape with a long cytoplasmic process reaching the intestinal lumen, but cells with round or oval shape were found in the CP4.5% and 0% groups. Frequencies of occurrence of neurotensin-immunoreactive cells in the CP18%, 9%, 4.5% and 0% groups were 42.4±3.3, 36.6±2.2, 30.8±2.6 and 25.4±3.8 (cell count per mucosal area: cells/mm2, mean±SD), respectively. There were significant differences in neurotensin-immunoreactive cell frequency between the control and lower CP level, 4.5% and 0%, groups. A significant correlation was found between frequency of occurrence of neurotensin-immunoreactive cells and daily protein intake. These results indicate that ingested protein is likely to be a potential signal for neurotensin production and secretion of N cells in the chicken ileum.

Half-life of Fructosyl-valine in the Plasma of Chicks.

Eighty 14-d-old single-comb White Leghorn male chicks were divided into 16 groups with five birds each. Fructosyl-valine, which is a valine-glucose-Amadori product, was intravenously (2,250 nmol/kg body weight) or orally (300 µmol/kg body weight) administered to chicks. Blood samples were collected 15, 30, 60, 120, 180, 360, 720 and 1440 min after administration. Plasma concentrations of fructosyl-valine were measured by using a liquid chromatography / mass spectrometry (LC/MS). The time course change in plasma fructosyl-valine concentration showed an exponential curve, as y=a+be-λt. The half-life of plasma fructosyl-valine was calculated by the following equation: (loge2)/λ. When fructosyl-valine was injected intravenously, the highest value for plasma fructosyl-valine concentration was observed 15 min after administration. When injected intravenously, the half-life of plasma fructosyl-valine was calculated to be 231 min. When fructosyl-valine was administered orally to chicks, the highest value for plasma fructosyl-valine concentration was observed 180 min after administration. When administered orally, the half-life of plasma fructosyl-valine was calculated to be 277 min. We conclude that the half-life of fructosyl-valine in plasma was approximately 4 h, which is longer than that of glycated tryptophan.

Nutritive evaluation of spent green and black tea leaf silages by in vitro gas production characteristics, ruminal degradability and post-ruminal digestibility assessed with inhibitory activity of their tannins.

Spent tea leaf contains high levels of crude protein, suggesting that it may be used as an alternative source for ruminant feeding. We assessed the nutritive characteristics of spent green tea leaf silage (GTS) and black tea leaf silages (BTS) in comparison with soybean meal (SBM) and alfalfa hay cube (AHC) using in vitro assay. The effects of tannin on the nutritive characteristics were also evaluated by adding polyethylene glycol (PEG) as a tannin-binding agent. The amount of gas production was greater for SBM, followed by AHC, GTS, and BTS. A significant improvement in gas production upon addition of PEG was observed only for BTS. Ruminal protein degradability and post-ruminal digestibility was higher for SBM, followed by AHC, GTS, and BTS. The presence of PEG significantly increased ruminal degradability and post-ruminal protein digestibility for GTS and BTS, but not for AHC. The increment of protein digestibility by PEG was much greater for BTS than for GTS, indicating that GTS tannins suppress protein digestibility slightly, whereas BTS tannins do so strongly. According to these results, GTS but not BTS has a potential as an alternative to AHC as a ruminant feedstuff.

Influence of long-term feeding of high-fat diet on quercetin and fat absorption from the small intestine in lymph duct-cannulated rats.

This study aimed to investigate the effect of dietary lipids and a long-term high-fat diet on lymphatic triglyceride and quercetin absorption in rats with a surgically implanted thoracic lymph cannula. Quercetin-3-O-ß-glucoside reduced the lymphatic triglyceride output from the intestines; this reduction was prominent among rats fed a high-fat diet.

Half-life of Glycated Tryptophan in the Plasma of Chickens.

Tryptophan, an essential amino acid, is enzymatically metabolized to two compounds, kynurenine and serotonin, and 95% of tryptophan is metabolized to kynurenine. As chickens have hyperglycemia and high temperature, tryptophan glycation occurs more easily in chickens than in mammals. Part of tryptophan is non-enzymatically converted to two types of glycated tryptophan, tryptophan-Amadori product and (1R, 3S)-1-(d-gluco-1, 2, 3, 4, 5-pentahydroxypentyl)-1,2,3,4-tetrahydro-ß-carboline-3-carboxylic acid (PHP-THßC). Although these compounds are detected in the plasma of chickens, information on the half-life of PHP-THßC in the blood circulation is limited. Therefore, the present study aimed to measure the half-life of plasma PHP-THßC in chickens. PHP-THßC (114 nmol/0.2 mL/70 g body weight) was intravenously administered to chickens via the wing vein, and blood samples were collected at 0, 15, 30, 60, 180, 360, 720, and 1440 min after administration. Plasma concentrations of PHP-THßC were measured by liquid chromatography-mass spectrometry. Plasma PHP-THßC reached to a peak concentration of 16.1 ßM at 30 min after administration, and then decreased rapidly to return to the physiological level (0 min) at 360 min after administration. The half-life of plasma PHP-THßC was calculated by non-linear regression analysis, and it was found to be 107 min. This study was the first to measure plasma half-life of glycated tryptophan.

Glucagon-like peptide-1 is co-localized with neurotensin in the chicken ileum.

Glucagon-like peptide (GLP)-1 and neurotensin (NT) are distributed throughout the chicken ileum. Here, we attempt to determine if GLP-1 and NT co-localize in the chicken ileum by using immunofluorescence, immunocytochemistry and in situ hybridization techniques. Three types of enteroendocrine cells, GLP-1+/NT+, GLP-1+/NT- and GLP-1-/NT+ cells, were detected in the mucosal epithelium by the double immunofluorescence method. The ratio of GLP-1+/NT+ cells at the crypts in the distal ileum was significantly higher than that in the proximal ileum. The ratios of the three cell types were similar along the crypt-villous axis in the proximal ileum but the percentage of GLP-1+/NT+ cells significantly decreased at the middle part of villi relative to crypts and the bottom part of villi in the distal ileum. Enteroendocrine cells that were immunoreactive to both GLP-1 and NT peptides and showed both proglucagon and NT precursor mRNA signals were found in the crypts of the distal ileum but not in the villous epithelium. The results from performing an immunocytochemical method with colloidal gold indicated that the GLP-1 content within GLP-1+/NT+ cell secretory granules decreased stepwise from the crypt to the middle part of the villus but the NT content in these granules increased in this direction. These findings reveal that the cells producing both GLP-1 and NT are mainly localized in the crypts of the chicken ileum but these endocrine cells specialize in NT-producing cells at the villous epithelium of the distal ileum.

Influence of Varying Dietary Protein Levels on Glycation of Albumin, Tryptophan and Valine in the Plasma of Chickens.

Glycation is a chemical reaction in which reducing sugars bind non-enzymatically to compounds containing amino groups. Avian species like chickens are hyperglycemic animals and have high body temperature compared to mammalian species, which enables avian species to accelerate the glycation of proteins and amino acids with glucose. Although varying dietary crude protein (CP) levels alter plasma concentrations of proteins and amino acids, the influence of varying CP levels on the glycation of plasma proteins and amino acids has not been studied so far. In the present study, therefore, glycation of albumin, tryptophan and valine in the plasma of chickens fed diets with varying CP levels (0, 10, 20, 40 and 60%) was examined. At the end of the experimental period, blood samples were collected and plasma concentrations of glycoalbumin, glycated tryptophan (tryptophan-Amadori product and (1R, 3S) - 1 - (D - gluco - 1, 2, 3, 4, 5 - pentahydroxypentyl) - 1, 2, 3, 4 - tetrahydro - ß - carboline - 3 - carboxylic acid (PHP-THßC)), and valine-Amadori product were measured. Although plasma albumin concentration was reduced along with the decrease in dietary CP levels from 20% to 0%, glycoalbumin in the plasma was increased under such dietary conditions. Similar increase in the ratios of tryptophan-Amadori product to tryptophan and valine-Amadori product to valine in the plasma of chickens fed a protein-free diet was observed. These results suggest that dietary protein deficiency might enhance the non-enzymatic glycation of plasma proteins and amino acids in chickens.

Influence of Trypsin-Digested Wheat Gluten Peptides with Different Molecular Size on Intestinal Absorption of Amino Acids in Chickens.

Although a lot of food-derived peptides have been applied for medical use and therapeutic nutrition, the function of feed-derived peptides on nutritional physiology in chickens has not been clarified so far. Our previous study revealed that wheat gluten digested by trypsin could enhance the absorption of amino acids from small intestine. In the present study, we studied the influence of trypsin-digested wheat gluten peptides with different molecular weight (MW) on the intestinal absorption of amino acids in chickens. Wheat gluten was digested by trypsin and fractionated by using the ultrafiltration membrane. Wheat gluten peptides were divided into 3 fractions with different MW; MW more than 10,000, MW 3,000-10,000 and MW less than 3,000. Phosphate buffered saline and whole wheat gluten digesta were used as negative and positive controls, respectively. All of wheat gluten peptides were mixed with 2.5 M glucose-10 mM amino acid solution and administrated into the crop with a stomach tube. At 20 min after oral administration, blood samples were taken from mesenteric vein. Plasma amino acid concentration was determined using an automatic amino acid analyzer. The peptide fraction with MW more than 10,000 increased the intestinal absorption of phenylalanine and proline. The peptide fraction with MW 3,000-10,000 increased the intestinal absorption of proline. These results suggest that wheat gluten peptide with high MW might have the potency to enhance the absorption of aromatic amino acids from small intestine of young chickens.

Fructosyl-Valine Orally Administrated to Chickens is Absorbed from Gastrointestinal Tract.

Amadori products are non-enzymatically formed by binding carbonyl groups and amino groups. Glycated amino acids generated by reacting amino acid and glucose are also in a group of Amadori products of which the transport and metabolism have been investigated mainly in mammals but not in avians. In the present study, therefore, we examined whether dietary fructosyl-valine, which is one of the glycated amino acids, orally administrated to chickens can be incorporated into blood or not. Fructosyl-valine was orally administrated to the chicken and blood samples were collected at 0, 20, 40, 60, 120 and 180 min after administration. Plasma concentration of fructosyl-valine was measured by using LC/MS. The plasma concentration of fructosyl-valine was increased by passing time from 0 to 180 min after administration, and no change was observed in the control group. Conclusively, it was clarified that fructosyl-valine orally administrated to the chicken could be absorbed from gastrointestinal tract and incorporated into blood.

Incorporation of Glycated-Tryptophan and -Valine into Various Cells Derived from Chicken Embryos.

Avian species including chickens are known to be hyperglycemic animals. Hyperglycemia promotes the glycation which at first forms Amadori products undergoing further complex reaction to form advanced glycation end products (AGEs). Our previous study revealed that AGEs derived from glucose and amino acids were predominantly incorporated into spleen, kidney and liver. However, it has not been elucidated whether Amadori products (glycated amino acids) can also be incorporated into cells or not. Therefore, in the present study, radioactive glycated-tryptophan and -valine were prepared and the incorporation of these glycated amino acids into various chicken embryonic cells was studied. Various embryonic cells prepared from muscle, liver, spleen and kidney of chicken embryos were incubated in Medium 199 supplemented with 14C-labeled glycated-tryptophan or -valine. After incubation, embryonic cells were well-rinsed and then the radioactivity incorporated into cells was measured. It was revealed that both glycated amino acids were incorporated into embryonic cells derived from muscle, liver, spleen and kidney. In muscular cells, the incorporation of glycated-tryptophan was higher than that of glycated-valine. On the other hand, in embryonic cells derived from liver and kidney, the amount of glycated-tryptophan incorporated into cells was almost the same to that of glycated-valine. In conclusion, it was supposed that both glycated-tryptophan and -valine could be incorporated into various cells derived from muscle, liver, spleen and kidney of chicken embryos and that the incorporation might have the organ specificity.

Assessment of Anti-nutritive Activity of Tannins in Tea By-products Based on In vitro Rumen Fermentation.

Nutritive values of green and black tea by-products and anti-nutritive activity of their tannins were evaluated in an in vitro rumen fermentation using various molecular weights of polyethylene glycols (PEG), polyvinyl pyrrolidone (PVP) and polyvinyl polypyrrolidone as tannin-binding agents. Significant improvement in gas production by addition of PEG4000, 6000 and 20000 and PVP was observed only from black tea by-product, but not from green tea by-product. All tannin binding agents increased NH3-N concentration from both green and black tea by-products in the fermentation medium, and the PEG6000 and 20000 showed relatively higher improvement in the NH3-N concentration. The PEG6000 and 20000 also improved in vitro organic matter digestibility and metabolizable energy contents of both tea by-products. It was concluded that high molecular PEG would be suitable to assess the suppressive activity of tannins in tea by-products by in vitro fermentation. Higher responses to gas production and NH3-N concentration from black tea by-product than green tea by-product due to PEG indicate that tannins in black tea by-product could suppress rumen fermentation more strongly than that in green tea by-product.

Fermentation Characteristics, Tannin Contents and In vitro Ruminal Degradation of Green Tea and Black Tea By-products Ensiled at Different Temperatures.

Green and black tea by-products, obtained from ready-made tea industry, were ensiled at 10°C, 20°C, and 30°C. Green tea by-product silage (GTS) and black tea by-product silage (BTS) were opened at 5, 10, 45 days after ensiling. Fermentation characteristics and nutrient composition, including tannins, were monitored and the silages on day 45 were subjected to in vitro ruminal fermentation to assess anti-nutritive effects of tannins using polyethylene glycol (PEG) as a tannin-binding agent. Results showed that the GTS and BTS silages were stable and fermented slightly when ensiled at 10°C. The GTS stored at 20°C and 30°C showed rapid pH decline and high acetic acid concentration. The BTS was fermented gradually with moderate change of pH and acid concentration. Acetic acid was the main acid product of fermentation in both GTS and BTS. The contents of total extractable phenolics and total extractable tannins in both silages were unaffected by storage temperatures, but condensed tannins in GTS were less when stored at high temperature. The GTS showed no PEG response on in vitro gas production, and revealed only a small increase by PEG on NH3-N concentration. Storage temperature of GTS did not affect the extent of PEG response to both gas production and NH3-N concentration. On the other hand, addition of PEG on BTS markedly increased both the gas production and NH3-N concentration at any ensiled temperature. It can be concluded that tannins in both GTS and BTS suppressed rumen fermentation, and tannins in GTS did more weakly than that in BTS. Ensiling temperature for both tea by-products did not affect the tannin's activity in the rumen.

Influences of protein ingestion on glucagon-like peptide (GLP)-1-immunoreactive endocrine cells in the chicken ileum.

Influences of a specific dietary nutrient on glucagon-like peptide (GLP)-1-containing cells in the chicken intestine are not yet clear. Significance of dietary protein level on GLP-1-containing cells in the chicken ileum was investigated. Chickens fed control or experimental diets of varying protein levels were examined using immunohistochemical and morphometrical techniques. We show that the protein ingestion had an impact on the activities of GLP-1-immunoreactive cells in the chicken ileum. Weight gains declined with decreasing dietary crude protein (CP) levels, but no significant differences were detected in the daily feed intake and villous height. GLP-1-immunoreactive cells with a round or oval shape were frequently observed in the lower CP level groups (4.5% and 0%). Frequencies of occurrence of GLP-1-immunoreactive cells were 41.1 ± 4.1, 38.5 ± 4, 34.8 ± 3.1 and 34.3 ± 3.7 (cells/mm(2) , mean ± SD) for dietary CP level of 18%, 9%, 4.5% and 0% groups, respectively and significant differences were recognized between the control and lower CP level groups (P<0.05). Multiple regression analysis indicated a significant correlation between the daily protein intake and frequencies of occurrence of GLP-1-immunoreactive cells. The protein ingestion is one of the signals that influence GLP-1-containing cells in the chicken small intestine.

Tissue distribution of albumin-glucose advanced glycation end products administrated to chickens.

Glycation (Maillard reaction) starts from non-enzymatic amino-carbonyl reaction binding carbonyl group of reducing sugars to amino group of amino acids. The amino-carbonyl reaction leads to the formation of the stable Amadori product, which ultimately forms advanced glycation end products (AGE). Acceleration of glycation during hyperglycemia increases the production and accumulation of AGE, which is implicated in the gradual development of diabetic complications in diabetes mellitus. Avian species offers the great advantage of providing animal models for diabetology because high blood glucose concentration should accelerate to generate high concentration of AGE. In the present study, radioactive AGE was prepared from chicken serum albumin and (14)C-glucose. Radioactive AGE was administrated to examine the tissue distribution of AGE in chickens. Administration of radioactive AGE into the circulation of chickens revealed that AGE accumulated into specific tissues, liver, spleen and kidney, which have the function for clearing exogenous substances and endogenous metabolic waste products.