Effect of chronic hypoxia on RAGE and its soluble forms in lungs and plasma of mice.

The receptor for advanced glycation end products (RAGE) is a multi-ligand receptor. Alternative splicing and enzymatic shedding produce soluble forms that protect against damage by ligands including Advanced Glycation End products (AGEs). A link between RAGE and oxygen levels is evident from studies showing RAGE-mediated injury following hyperoxia. The effect of hypoxia on pulmonary RAGE expression and circulating sRAGE levels is however unknown. Therefore mice were exposed to chronic hypoxia for 21 d and expression of RAGE, sheddases in lungs and circulating sRAGE were determined. In addition, accumulation of AGEs in lungs and expression of the AGE detoxifying enzyme GLO1 and receptors were evaluated. In lung tissue gene expression of total RAGE, variants 1 and 3 were elevated in mice exposed to hypoxia, whereas mRAGE and sRAGE protein levels were decreased. In the hypoxic group plasma sRAGE levels were enhanced. Although the levels of pro-ADAM10 were elevated in lungs of hypoxia exposed mice, the relative amount of the active form was decreased and gelatinase activity unaffected. In the lungs, the RAGE ligand HMGB1 was decreased and of the AGEs, only LW-1 was increased by chronic hypoxia. Gene expression of AGE receptors 2 and 3 was significantly upregulated. Chronic hypoxia is associated with downregulation of pulmonary RAGE protein levels, but a relative increase in sRAGE. These alterations might be part of the adaptive and protective response mechanism to chronic hypoxia and are not associated with AGE formation except for the fluorophore LW-1 which emerges as a novel marker of tissue hypoxia.

End-stage renal disease and diabetes catalyze the formation of a pentose-derived crosslink from aging human collagen.

Structure elucidation of a specific fluorophore from the aging extracellular matrix revealed the presence of a protein crosslink formed through nonenzymatic glycosylation of lysine and arginine residues. The unexpected finding that a pentose instead of a hexose is involved in the crosslinking process suggested that the crosslink, named pentosidine, might provide insight into abnormalities of pentose metabolism in aging and disease. This hypothesis was investigated by quantitating pentosidine in hydrolysates of 103 human skin specimens obtained randomly at autopsy. Pentosidine level was found to increase exponentially from 5 to 75 pmol/mg collagen over lifespan (r = 0.86, P less than 0.001). A three- to tenfold increase was noted in insulin-dependent diabetic and nondiabetic subjects with severe end-stage renal disease requiring hemodialysis (P less than 0.001). Moderately elevated levels were also noted in some very old subjects, some subjects with non-insulin dependent diabetes, and two subjects with cystic fibrosis and diabetes. The cause of the abnormal pentose metabolism in these conditions is unknown but may relate to hemolysis, impaired pentose excretion, cellular stress, and accelerated breakdown of ribonucleotides. Thus, pentosidine emerges as a useful tool for assessment of previously unrecognized disorders of pentose metabolism in aging and disease. Its presence in red blood cells and plasma proteins suggests that it might be used as a measure of integrated pentosemia in analogy to glycohemoglobin for the assessment of cumulative glycemia.

Pentosidine formation in skin correlates with severity of complications in individuals with long-standing IDDM.

Pentosidine is an advanced glycosylation end product and protein cross-link that results from the reaction of pentoses with proteins. Recent data indicate that long-term glycation of proteins with glucose also leads to pentosidine formation through sugar fragmentation. In this study, the relationship between the severity of diabetic complications and pentosidine formation was investigated in collagen from skin-punch biopsies from 25 nondiabetic control subjects and 41 IDDM patients with diabetes duration greater than 17 yr. Pentosidine was significantly elevated in all IDDM patients versus control subjects (P less than 0.0001). It correlated strongly with age (P less than 0.0001) and weakly with duration (P less than 0.082). Age-adjusted pentosidine levels were highest in grade 2 (severe) versus grade 1 and 0 complication in all four parameters tested (retinopathy, proteinuria, arterial stiffness, and joint stiffness). Significant differences were found for retinopathy (P less than 0.014) and joint stiffness (P less than 0.041). The highest degree of association was with the cumulative grade of individual complication (P less than 0.005), determined by summing indexes of all four parameters. Pentosidine also was significantly elevated in the serum of IDDM patients compared with control subjects (P less than 0.0001), but levels were not significantly correlated with age, diabetes duration, complication, or skin collagen pentosidine (P greater than 0.05). A high correlation between pentosidine levels and long-wave collagen-linked fluorescence also was observed, suggesting that pentosidine is a generalized marker of accelerated tissue modification by the advanced glycosylation/Maillard reaction, which is enhanced in IDDM patients with severe complications.

Chromatographic quantitation of plasma and erythrocyte pentosidine in diabetic and uremic subjects.

Pentosidine is a fluorescent advanced Maillard/glycosylation product and protein cross-link present in elevated amounts in skin from diabetic and uremic subjects. A high-performance liquid chromatographic (HPLC) assay was developed to quantitate pentosidine in plasma and erythrocytes and other tissue proteins with low levels of pentosidine. High protein content and presence of basic amino acids and O2 during acid hydrolysis led to the formation of fluorescent artifacts that could be separated from true pentosidine through combined reverse-phase ion-exchange HPLC. No true pentosidine was formed during acid hydrolysis of ribated protein, suggesting that Amadori products do not generate artifactual pentosidine during hydrolysis. With the combined reverse-phase ion-exchange chromatographic assay, we found a 2.5-fold (P less than 0.001) and a 23-fold (P less than 0.001) elevation of mean +/- SD plasma protein pentosidine in diabetic (2.4 +/- 1.2 pmol/mg) and uremic (21.5 +/- 10.8 pmol/mg) subjects compared with healthy (0.95 +/- 0.33 pmol/mg) subjects. Pentosidine in hemolysate was normal in diabetes but dramatically elevated in uremia (0.6 +/- 0.4 pmol/mg hemoglobin, P less than 0.001). Although the precise nature of the pentosidine precursor sugar is unknown, plasma pentosidine may be a useful marker for monitoring the biochemical efficacy of trials with aminoguanidine or other treatment modalities. Furthermore, pentosidine in plasma proteins may act as a signal for advanced glycosylation end product-mediated receptor uptake by macrophages and other cells and contribute to accelerated atherosclerosis in diabetes and uremia.

Tissue-specific effects of aldose reductase inhibition on fluorescence and cross-linking of extracellular matrix in chronic galactosemia. Relationship to pentosidine cross-links.

Chronic experimental hyperglycemia mediated by galactose has been shown to induce browning and cross-linking of rat tail tendon collagen that could be duplicated in vitro by nonenzymatic galactosylation. To investigate the nature of these changes, Sprague-Dawley rats were placed on a 33% galactose diet without and with sorbinil for 6 and 12 mo. Collagen-linked fluorescence and pentosidine cross-links increased with age and galactosemia in tail tendons (P less than 0.001) and skin but were essentially unresponsive to aldose reductase inhibition (ARI). In contrast, tendon breaking time in urea, a likely parameter of cross-linking, was markedly improved (P less than 0.001) by ARI. Fluorescence that was inhibited by sorbinil treatment was increased in pepsin and proteinase K digest of aortic tissue from galactosemic rats (P less than 0.001), but impaired enzymatic digestibility was not observed. Systolic blood pressure as potential consequence of aortic stiffening was not increased in galactosemia. These data suggest that fluorescence in skin and tendon might be in part due to advanced glycosylation and pentosidine formation because these were not decreased by ARI. However, they also suggest that nonfluorescent cross-links may also be forming because, in contrast to fluorescence, tail tendon breaking time was partly corrected by ARI. Thus, it appears that extracellular matrix changes in chronic galactosemia are complex, being partly attributable to advanced glycosylation and partly to polyol-pathway activation.

The mechanism of collagen cross-linking in diabetes: a puzzle nearing resolution.

Considerable interest has been focused in recent years on the mechanism of collagen cross-linking by high glucose in vitro and in vivo. Experiments in both diabetic humans and in animals have shown that over time collagen becomes less soluble, less digestible by collagenase, more stable to heat-induced denaturation, and more glycated. In addition, collagen becomes more modified by advanced products of the Maillard reaction, i.e., immunoreactive advanced glycation end products and the glycoxidation markers carboxymethyllysine and pentosidine. Mechanistic studies have shown that collagen cross-linking in vitro can be uncoupled from glycation by the use of antioxidants and chelating agents. Experiments in the authors' laboratory revealed that approximately 50% of carboxymethyllysine formed in vitro originates from pathways other than oxidation of Amadori products, i.e., most likely the oxidation of Schiff base-linked glucose. In addition, the increase in thermal stability of rat tail tendons exposed to high glucose in vitro or in vivo was found to strongly depend on H2O2 formation. The final missing piece of the puzzle is that of the structure of the major cross-link. We speculate that it is a nonfluorescent nonultraviolet active cross-link between two lysine residues, which includes a fragmentation product of glucose linked in a nonreducible bond labile to both strong acids and bases.

Collagen browning and cross-linking are increased in chronic experimental hyperglycemia. Relevance to diabetes and aging.

Diabetes and aging are associated with an increase in collagen-linked fluorescence and cross-linking that can be duplicated by incubating collagen with glucose. We have tested the hypothesis that browning and cross-linking can occur in vivo in rats by feeding them a diet containing 33% galactose. No significant increase in tail tendon browning or cross-linking, measured by tail tendon breaking time in urea, was observed after 3 mo of galactosemia. After 12 mo, however, twofold increases in tendon breaking time and collagen-linked chromophores absorbing greater than 300 nm and fluorophores at 430 nm (excitation 355 nm, P less than .001) analogous to those of diabetic and aging individuals were observed. The observed changes in collagen are attributed to the advanced Maillard (nonenzymatic glycosylation) reaction based on circumstantial evidence. With this premise, our data suggest that chronic galactosemia should be a powerful tool for investigating the role of the advanced Maillard reaction in complications of diabetes and aging.

Evidence of a glycemic threshold for the formation of pentosidine in diabetic dog lens but not in collagen.

The relationship between long-term glycemic control and the advanced Maillard reaction was investigated in dura mater collagen and lens proteins from dogs that were diabetic for 5 years. Diabetic dogs were assigned prospectively to good, moderate, and poor glycemic control and maintained by insulin. Biochemical changes were determined at study exit. Mean levels of collagen digestibility by pepsin decreased (NS) whereas collagen glycation (P < 0.001), pentosidine cross-links (P < 0.001), and collagen fluorescence (P = 0.02) increased with increasing mean HbA1 values. Similarly, mean levels of lens crystallin glycation (P < 0.001), fluorescence (P < 0.001), and the specific advanced lens Maillard product 1 (LM-1) (P < 0.001) and pentosidine (P < 0.005) increased significantly with poorer glycemic control. Statistical analysis revealed very high Spearman correlation coefficients between collagen and lens changes. Whereas pentosidine cross-links were significantly elevated in collagen from diabetic dogs with moderate levels of HbA1 (i.e., 8.0 +/- 0.4%), lens pentosidine levels were normal in this group and were elevated (P < 0.001) only in the animals with poor glycemic control (HbA1 = 9.7 +/- 0.6%). Thus, whereas protein glycation and advanced glycation in the extracellular matrix and in the lens are generally related to the level of glycemic control, there is evidence for a tissue-specific glycemic threshold for pentosidine formation, i.e., glycoxidation, in the lens. This threshold may be in part linked to a dramatic acceleration in crystallin glycation with HbA1 values of > 8.0% and/or a loss of lens membrane permeability. This study provides support at the molecular level for the growing concept that glycemic thresholds may be involved in the development of some of the complications in diabetes.

Maillard reaction-mediated molecular damage to extracellular matrix and other tissue proteins in diabetes, aging, and uremia.

Recent progress in structure elucidation of products of the advanced Maillard reaction now allows probing specifically for the role of this reaction in the pathogenesis of age- and diabetes-related complications. Pyrraline is a glucose-derived advanced glycation end product against which polyclonal and monoclonal antibodies have been raised. Immunohistochemical localization studies revealed that pyrraline is found predominantly in the sclerosed extracellular matrix of glomerular and arteriolar renal tissues from both diabetic and aged nondiabetic individuals. Pentosidine and carboxymethyllysine are Maillard end products derived from both glucose and ascorbate. In addition, pentosidine can be formed from several other sugars under oxidative conditions, and in vitro studies suggest that a common intermediate involving a pentose is a necessary precursor molecule. The highest levels of these advanced Maillard products are generally found in the extracellular matrix, but these products are also present in lens proteins and in proteins with a fast turnover such as plasma proteins. Diabetes, and especially uremia, greatly catalyzes pentosidine formation. Both conditions are characterized by accelerated cataractogenesis, atherosclerosis, and neuropathy, suggesting that molecular damage by advanced Maillard reaction products may be a common mechanism in their development.

Skin collagen glycation, glycoxidation, and crosslinking are lower in subjects with long-term intensive versus conventional therapy of type 1 diabetes: relevance of glycated collagen products versus HbA1c as markers of diabetic complications. DCCT Skin Collagen Ancillary Study Group. Diabetes Control and Complications Trial.

The relationships between long-term intensive control of glycemia and indicators of skin collagen glycation (furosine), glycoxidation (pentosidine and N(epsilon)-[carboxymethyl]-lysine [CML]), and crosslinking (acid and pepsin solubility) were examined in 216 patients with type 1 diabetes from the primary prevention and secondary intervention cohorts of the Diabetes Control and Complications Trial. By comparison with conventional treatment, 5 years of intensive treatment was associated with 30-32% lower furosine, 9% lower pentosidine, 9-13% lower CML, 24% higher acid-soluble collagen, and 50% higher pepsin-soluble collagen. All of these differences were statistically significant in the subjects of the primary prevention cohort (P < 0.006-0.001) and also of the secondary intervention cohort (P < 0.015-0.001) with the exception of CML and acid-soluble collagen. Age- and duration-adjusted collagen variables were significantly associated with the HbA1c value nearest the biopsy and with cumulative prior HbA1c values. Multiple logistic regression analyses with six nonredundant collagen parameters as independent variables and various expressions of retinopathy, nephropathy, and neuropathy outcomes as dependent variables showed that the complications were significantly associated with the full set of collagen variables. Surprisingly, the percentage of total variance (R2) in complications explained by the collagen variables ranged from 19 to 36% with the intensive treatment and from 14 to 51% with conventional treatment. These associations generally remained significant even after adjustment for HbA1c, and, most unexpectedly, in conventionally treated subjects, glycated collagen was the parameter most consistently associated with diabetic complications. Continued monitoring of these subjects may determine whether glycation products in the skin, and especially the early Amadori product (furosine), have the potential to be predictors of the future risk of developing complications, and perhaps be even better predictors than glycated hemoglobin (HbA1c).

Ageing promotes the increase of early glycation Amadori product as assessed by epsilon-N-(2-furoylmethyl)-L-lysine (furosine) levels in rodent skin collagen. The relationship to dietary restriction and glycoxidation.

Glucose has been implicated in the aging process by its ability to react nonenzymatically with long-lived proteins like collagen to produce advanced glycosylated end-products (AGEs). In the initial phase of this reaction, referred to as glycation, glucose reacts with the free amino group of proteins resulting in Schiff base formation followed by rearrangement to an Amadori product. Since the Amadori product is transient due to its conversion to other products as well as its reversibility to the initial products, glycation as an age-related marker in collagen has questionable significance. In human studies, glycation of collagen has been found to increase modestly with age. In rodent studies, results are conflicting due to differences in methodology. Thus, it has been concluded that collagen glycation either does not vary or increases modestly with age. In the present study, a C8 HPLC column was used to measure Amadori product formation as the acid-hydrolyzed breakdown product furosine in the skin of rats and mice. Surprisingly, levels were found to increase at a rapid rate during aging of rodents. Impurity of the furosine peak from the use of crude acid-hydrolyzed skin samples was ruled-out because reductive properties and spectroscopic profiles matched those previously described for furosine. In the present study, glycemia was found important in furosine formation as shown by the glycation lowering effects of dietary restriction on collagen. Decreased collagen turnover probably plays a substantial role in explaining the age-related increase in furosine levels in rodent skin collagen.

Cell-associated pentosidine as a marker of aging in human diploid cells in vitro and in vivo.

Cellular aging is characterized by alterations at both the morphological and molecular levels, some of which are decreased mitotic rate, increased cytoplasmic vacuolization, and changes in intrinsic cellular constituents (Stanulis-Praeger, 1987. Mech. Ageing Dev. 38, 1-48). In the present investigation, glycoxidation is studied as a marker for cellular aging by measuring cell-associated pentosidine levels in human skin fibroblasts as a function of replicative life span and in human peripheral blood T lymphocytes as a function of chronological age. Fibroblasts were isolated from culture by detachment/centrifugation while lymphocytes were isolated from blood by a Ficoll-Paque/Lympho-Kwik T-Cell Prep technique. Pentosidine levels were measured in acid-hydrolyzed cell pellet suspensions by high-pressure liquid chromatography. Results show that pentosidine was detected in early and late cultured reticular and papillary fibroblasts. Pentosidine, expressed as either protein, DNA, or cell number, significantly (P < 0.0006) increased with in vitro passage and was significantly (P < 0.01) related to cell proliferation as measured by cell density and cell doublings per day during culture. Cell-associated pentosidine was measured in T lymphocytes isolated from healthy, diabetic, and uremic individuals. In healthy controls, levels significantly (P < 0.0003) increased with age. In uremic individuals, a large variation was observed with many values above the 95% confidence intervals determined for controls. Since a previous study showed that plasma pentosidine in healthy subjects does not increase with age, these results suggest that cellular turnover perhaps coupled to a deterioration in cellular anti-glycoxidation defensive mechanisms play a substantial role in explaining increased pentosidine concentrations during cellular aging.

Age-related association of tail tendon break time with tissue pentosidine in DBA/2 vs C57BL/6 mice: the effect of dietary restriction.

In recent years, there has been a growing interest in the development of a panel of biomarkers useful in the evaluation of interventions on aging processes. An ideal marker should change with age, be related to species longevity, and respond to the effects of dietary restriction, which is the only intervention currently known to increase species longevity. In the present study; we compared parameters of collagen aging (i.e., tail tendon break time [TBT] and the glycoxidation product pentosidine) in tendon, ear, and skin of two species of rodents with different life spans: the shorter-lived DBA/2 versus the longer-lived C57BL/6 mouse strain. Both TBT and tissue pentosidine significantly increased with age in both strains of mice. The rate of increase for TBT And pentosidine occurred faster for the DBA/2 compared with the C57BL/6 strain. Dietary restriction significantly inhibited the age-related increase of TBT and pentosidine formation rte in DBA/2 mice. In C57BL/6 mice, the age-related increase of TBT was significantly inhibited by dietary restriction. However, except for tendon at 24 months, pentosidine level was not affected by dietary restriction. These studies show that the rate of collagen aging, as reflected by TBT and glycoxidation, increases proportionally with age, and that these rate increases are related to longevity in two strains of mice. Pentosidine can be monitored with age just as well in a piece of easily accessible ear tissue as in skin or tendon. Thus, pentosidine is expected to be a useful and easily measurable noninvasive marker in future intervention studies on aging.

Effect of chronic aminoguanidine treatment on age-related glycation, glycoxidation, and collagen cross-linking in the Fischer 344 rat.

Aminoguanidine (AG) is an inhibitor of protein modification by the advanced Maillard reaction. We evaluated its effects in preventing age-related collagen cross-linking, glycation, and glycoxidation in Fischer 344 rats by administering the drug in their drinking water at 1 g/l from the time they were 6 months until they were 24 months of age. Body weight and food and water consumption were consistently recorded throughout the study. Plasma glucose was measured by the glucose oxidase method, and collagen cross-linking was assessed by tail tendon break time (TBT) in urea. Glycation (furosine) and glycoxidation (pentosidine and carboxymethyllysine) were assessed by high-performance liquid chromatography in acid hydrolysates of skin and tendon collagen. Water consumption dramatically increased (p <.0001) after 20 months of age and was accelerated in the control versus AG-treated rats (p <.0001). Plasma glucose increased approximately 20% at age 19 months in both groups (p <.0001). TBT, glycation, and glycoxidation all increased significantly (p <.0001) with age. However, except for a modest decrease of TBT at all ages that approached significance (p =.077), AG had no effect on collagen glycation or glycoxidation. These results are important because they suggest that alpha,beta-dicarbonyl compounds that can be trapped by aminoguanidine do not play a major role in collagen aging in the rat. Instead, post-Amadori pathways involving oxidative or nonoxidative fragmentation of the Amadori product emerge as the more likely mechanism of collagen cross-linking in aging.

A syndrome of molybdenosis, copper deficiency, and type 2 diabetes in the moose population of south-west Sweden.

Since the mid-1980s, a 'mysterious' disease has been afflicting the moose (Alces alces L.) population of south-western Sweden. Molybdenosis combined with secondary copper deficiency syndrome has been suggested as the cause of the clinical signs and of necropsy findings, supported by trace element analysis. Copper deficiency has long been associated with disturbed carbohydrate metabolism and also with oxidative stress. When testing the oxidative stress hypothesis, we found increased concentrations of the glycoxidation products pentosidine and carboxymethyl-lysine (CML), both in plasma proteins and in renal tissue, when compared with control values. The concentration of glycated lysine (furosine), a marker of hyperglycaemia, was also increased. These data, together with elevated insulin levels in affected moose, strongly suggest that they are suffering from an environmentally-induced, non-insulin-dependent type 2 diabetes.

The effects of sorghum tannin and methionine level on the performance of laying hens maintained in two temperature environments.

At the conclusion of a 31-day recovery period following a previous sorghum feeding trial (Sell et al., 1983), 128 White Leghorn hens were used in a subsequent 44-day study to investigate the effects of methionine supplementation of sorghum-soybean meal diets on laying hen performance. The hens were arrayed into four nutritional treatments in each of two constant environments (22 and 28 C) based on their egg production during the last 10 days of the recovery period within a previous treatment and environment so that equal numbers of hens from the previous treatments were represented in the new treatments. Nutritional treatments consisted of either low (RS610) or high (savanna) tannin sorghum-soybean meal diets containing 13% protein and supplemented with either .2% DL-methionine (Met) or an isonitrogenous level of L-glutamic acid (Glu). With both high (HTS) and low tannin sorghum (LTS) diets, Met supplementation resulted in greater egg production, egg weight, feed intake, and less hen weight loss as compared with Glu addition, but with the exception of hen weight loss, the magnitude of the response was much greater with HTS. Hens fed the HTS diet supplemented with Glu showed greatly reduced egg production and feed efficiency as compared with those fed LTS similarly supplemented. In contrast, little difference in egg production and feed efficiency was observed between hens fed HTS and LTS diets supplemented with Met. All parameters studied except hen weight appeared to be reduced slightly by the higher temperature.

The effects of sorghum tannin and protein level on the performance of laying hens maintained in two temperature environments.

A study was conducted to investigate the effect of sorghum grain tannins on the performance of laying hens. A total of 144 White Leghorn hens were arrayed according to egg production during a 10-day preexperimental period into eight groups consisting of four nutritional treatments in each of two constant temperature environments (22 and 28 C). The nutritional treatments consisted of low (RS610) and high (Savanna) tannin sorghum grains compared isonitrogenously within a protein level at two protein levels (11.5 and 14.5%) in sorghum-soybean meal diets. These diets were fed for an experimental period of 6 weeks. Egg production and feed efficiency were reduced and weight loss was increased by 11.5% protein as compared with 14.5% protein. Tannin significantly reduced egg production and feed efficiency at both protein levels while egg specific gravity and shell thickness were adversely affected only at the lower level of protein. Egg weight and weight loss of hens were not affected by tannin. Except for weight loss, all parameters were slightly reduced by the higher temperature. At the end of the experiment, all hens were placed on a commercial laying ration for a 31-day period. Recovery from the treatment effects was evidenced by the similar egg production, body weight, egg weight, and shell characteristics among the previous treatments at the end of this period.

Methionine-cystine interrelationships in chicks and rats fed diets containing suboptimal levels of methionine.

Studies were continued with both chicks and rats on the previously reported (featherston and Rogler, 1978) growth depression observed when a crystalline amino acid diet adequate in all nonsulfur-containing amino acids and containing .2% DL-methionine and .2% L-cystine was supplemented with an additional .2% L-cystine. In the present investigation, an average growth depression of 37% was observed in chicks fed a diet containing .4% L-cystine and .2% DL-methionine as compared with chicks fed a diet containing .2% L-cystine and the same level of methionine. A comparable depression was noted when cystine was replaced by L-cysteine. In contrast, an additional .2% of another amino acid (L-tryptophan) did not depress growth. When the high and low cystine diets were meal-fed, results obtained were similar to those observed with ad libitum feeding, but the magnitude of the difference was reduced. Foot-pad lesions, similar to those described in turkey poults fed methionine-deficient diets, were observed in two experiments. Factorial analysis of plasma amino acids indicated that the total of all amino acids analyzed was significantly (P < .05) higher in chicks fed the lower cystine or cysteine diets. Plasma methionine was slightly lower (P < .10) but plasma cystine was significantly (P < .05) higher in chicks fed the cystine or cysteine supplemented diets. A study with .2 and .4% L-cystine and 200, 600, and 2000 mg/kg of choline (factorial design) demonstrated a significant (P < .05) depression in weight gain due to the added L-cystine, no significant (P < .05) differences due to choline, and the absence of a cystine-choline interaction. No detrimental effects were observed when weanling rats were fed a crystalline amino acid diet containing .1% DL-methionine and .2% L-cystine supplemented with additional L-cystine.

Influence of dietary cystine on intestinal absorption and tissue distribution of methionine in the chick.

Studies were conducted on intestinal absorption and tissue distribution of methionine by chicks fed a crystalline amino acid diet containing .2% DL-methionine and .2 or .4% L-cystine or L-cysteine. Chicks fed the higher level of cystine or cysteine had previously been shown to exhibit depressed growth as opposed to chicks fed the lower level of these amino acids. In two experiments, 35S radioactivity of whole plasma and the protein and supernatant fractions were compared among chicks fed the different treatments at 30 or 60 min after oral intubation of L-[35S]-methionine into the crops. When the data were analyzed factorially, a significant level effect was noted for whole plasma and the protein and supernatant fractions, all of which were higher for chicks fed .2% L-cystine than those fed .4% L-cystine. Results with chicks fed the two levels of L-cysteine generally agreed with those of chicks fed L-cystine, although the differences were not as great. The labeled methionine was also added to diets containing .2 or .4% L-cystine of meal-fed chicks. Higher levels of radioactivity were observed in the liver but not in muscle or plasma of chicks fed the lower level of cystine at 7 hr after oral consumption of the isotope. No significant differences in liver, muscle, or plasma radioactivity were noted in a fourth experiment between chicks fed .2 or .4% L-cystine at 24 hr after intraperitoneal injection of L-[35S]-methionine. In vitro studies showed no differences in methionine accumulation by isolated intestinal segments incubated with three levels of cystine.