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Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions

Excess Dietary L-Cysteine, but Not L-Cystine, Is Lethal for Chicks but Not for Rats or Pigs

¹ Department of Animal Sciences and Division of Nutritional Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801 and ² Ajinomoto, Institute of Life Sciences, Kawasaki, Japan

^* To whom correspondence should be addressed. E-mail: dhbaker{at}uiuc.edu.

	ABSTRACT

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
A comparative species investigation of the relative pharmacologic effects of sulfur amino acids was conducted using young chicks, rats, and pigs. Ingestion of excess Met, Cys, or Cys-Cys supplemented at 2.5-, 5.0-, 7.5-, or 10 times the dietary requirement in a corn-soybean meal diet depressed chick growth to varying degrees. Strikingly, ingestion of excess Cys at 30 g/kg Cys (7.5-times the dietary requirement) caused a chick mortality rate of 50% after only 5 d of feeding. Growth was restored and chick mortality was reduced by supplementing diets containing 25 g/kg excess Cys with KHCO₃ at 10 g/kg. Additionally, mortality was prevented by supplementing the drinking water of chicks receiving 25 g/kg supplemental Cys with H₂O₂ (0.05% final concentration). After young rats and pigs consumed excess Cys or Cys-Cys up to 40 g/kg for 14 d, weight gain was severely depressed, but we observed no mortality. An excess of dietary Cys-Cys 48 g/kg caused some mortality in rats. Pigs exhibited rapid recovery from growth-depressing excesses of Cys or Cys-Cys. These results lend credence to the acute toxic effects associated with the ingestion of excess sulfur amino acids and highlight the potential for excess dietary cyst(e)ine to be more pernicious than Met in certain species.

	Introduction

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

The SAA perform myriad functions in nutrition not limited to protein synthesis and structure, transfer of methyl groups, and as precursors of glutathione, taurine, and coenzyme A. The highly integrated nature of SAA in intermediary metabolism potentiates differential utilization when ingested at levels above dietary requirements. Although excess Met is known to cause greater growth depression than other amino acids (5–7), a dearth of information exists regarding ingestion of excessive amounts of Cys or Cys-Cys in animal models. The literature alludes to noxious effects of ingesting Cys (5), but comprehensive investigations do not exist.

Both the reduced (Cys) and oxidized (Cys-Cys) forms of cyst(e)ine support animal growth equally when provided in a cyst(e)ine-deficient and Met-adequate diet (8). However, to our knowledge, no studies have simultaneously compared the upper dose levels of these 2 sources of Cys. Our primary objective in this study was to compare the relative effects of ingesting excess SAA, particularly Cys and Cys-Cys, in young chicks, rats, and pigs. We also searched for ameliorative treatments to counteract Cys toxicity. The excess dietary cyst(e)ine concentrations used in our studies were above typical human intakes, but the data may be useful in defining upper tolerance levels for Cys and Cys-Cys used as over-the-counter nonprescription products.

	Materials and Methods

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
Chick experiments

All experimental procedures were approved by the University of Illinois Animal Care and Use Committee. Studies were conducted using male chicks (New Hampshire males crossed with Columbian females) obtained from the University of Illinois Poultry Farm. Chicks were housed in thermostatically controlled starter batteries with raised-wire flooring in an environmentally controlled room with continuous lighting. From hatch to d 7 posthatch, chicks were fed a 230 g/kg crude protein (CP) corn-soybean meal starter diet adequate in all dietary nutrients (9). Following an overnight fast, chicks were weighed, wing-banded, and randomized to dietary treatments on d 8 such that mean initial pen weights and weight distributions were similar across treatments.

The experimental corn-soybean meal diet (Table 1) was formulated to be nutritionally adequate (9) for this age chick, and was fed for a 9-d period (d 8–17 posthatch) during each chick experiment. Experimental diets and tap water were freely available to chicks at all times unless otherwise specified. Comparison of SAA was done using L-Met, L-Cys, and L-Cys-Cys in all studies. Body weight (BW) of individual chicks and pen food intakes were measured at the termination of each experiment. Weight gain, food intake, and food efficiency (i.e., gain:food ratio) were calculated for each replicate pen.

    Experiment 2. The objective was to test the ameliorative effect of KHCO₃ on chick growth depression due to ingestion of excess Cys. Dietary additions included 25 g/kg Cys or this plus 0, 2.5, 5.0, 7.5, or 10.0 g/kg KHCO₃. Each dietary treatment (6 total) was fed to 4 replicate pens of 3 chicks (87 g mean initial BW). Upon termination of the experiment, chicks were killed via CO₂ asphyxiation and blood was collected for analysis of pH, lactate, partial pressure of carbon dioxide, partial pressure of oxygen, bicarbonate, total carbon dioxide, base excess, and saturation of oxygen according to previously described procedures (12).

    Experiment 3. The objective was to determine whether H₂O₂ supplied in the drinking water could ameliorate the pharmacologic effects of ingesting excess Cys. Diets contained supplemental Cys at 0 or 25 g/kg, and chicks received purified (deionized) drinking water supplemented with either 0 or 0.05% H₂O₂ (v:v, final concentration). The H₂O₂ solution was freshly prepared before the experiment began, and was stored in an opaque container to avoid degradation by ultraviolet light. Glass waterers (wrapped with aluminum foil) were cleaned daily and fresh H₂O₂ solution was added after each cleaning. Treatments (4 total) were supplied to 3 replicate pens of 3 chicks (93 g mean initial BW). Water intake was monitored daily.

    Miscellaneous chick experiments. Four replicate pens of 3 chicks were fed diets based on the standard corn-soybean meal basal (Table 1) to address various tangential objectives pertinent to Cys toxicity. Growth performance of chicks receiving 25 g/kg excess L-Cys or an isomolar level of L-Cys · HCl was evaluated over a 9-d feeding period to investigate whether toxicity was related to Cys source. To determine the effect of intraperitoneal Cys injections, a solution of L-Cys was prepared in physiological saline. Chicks received a 1-mL injection of saline (control), or 300 or 600 mg Cys/kg BW (Cys dissolved in saline) after being acclimated to the corn-soybean meal diet. These Cys concentrations were used to confirm previous results from our laboratory (unpublished data). Growth performance and general health status of chicks were monitored for 5 d postinjection.

To investigate the effect of a bolus dose of Cys (i.e., mimicking ingestion of a Cys capsule by humans), chicks were acclimated to the standard corn-soybean meal diet for 10 d posthatch. Then, food-deprived chicks were oral-gavaged (i.e., crop intubated) with distilled water (control) or 600 mg Cys (dissolved in distilled water) in 6–1 mL doses over a 30-min period. This quantity of Cys (4.0 g/kg BW) was previously calculated to be the mean daily Cys intake of chicks consuming a corn-soybean meal diet with 25 g/kg supplemental Cys. Chick weight gain, food intake, and health status were monitored for the subsequent 24-h period following Cys gavage.

Rat experiments

All experimental procedures were approved by Ajinomoto's Institutional Animal Care and Use Committee. Male Fischer rats (F-344; 5–6 wk old) purchased from Charles River Japan were housed individually in stainless steel metabolism cages (wire mesh bottom) in a controlled (23°C; 12-h light and 12-h dark cycle) environment. Rats were acclimated to the casein-based experimental diet containing 5.6 g/kg Met and 3.7 g/kg cyst(e)ine for 4 d prior to initiation of dietary treatments. The basal diet (Table 1) was based on an AIN-93G formulation, and was therefore nutritionally adequate. Experimental diets and tap water were freely available to rats throughout each experiment. Comparison of cyst(e)ine sources was done using L-Cys and L-Cys-Cys in all studies. Doses of excess dietary Cys and Cys-Cys ranged from 1.2 g/kg to 7.2 g/kg in most rat studies, and these concentrations of excess cyst(e)ine represent estimated 4- and 24-fold increases, respectively, above the cyst(e)ine requirement of young rats.

    Experiments 1 and 2. Two experiments were conducted to characterize the effects of ingesting excess Cys (rat Expt. 1) or Cys-Cys (rat Expt. 2) on growth over a 14-d feeding period. Dietary additions of Cys or Cys-Cys at 0, 3, 6, 12, 24, or 48 g/kg were made at the expense of cornstarch. Each dietary treatment (6 total per experiment) was fed to 6 rats. The mean initial BW of rats in Expt. 1 and 2 were 101 and 121 g, respectively. Weight gain was calculated for individual rats using cumulative data from the 14-d experiments.

    Experiment 3. Dietary additions of Cys or Cys-Cys at 0, 24, 48, or 72 g/kg were made at the expense of cornstarch. Each dietary treatment (7 total) was fed to 6 rats (mean initial BW = 115 g) over a 5-d period. The 7.2 g/kg doses of Cys and Cys-Cys in the diet approximate a daily intake of 3.0 g/kg BW. Weight gain was calculated for individual rats using cumulative data from the 5-d experiment.

    Experiment 4. This experiment was the accumulation of 6 independent studies all using the casein-based basal diet (Table 1). Dietary additions of Cys or Cys-Cys at 48 or 72 g/kg were made at the expense of cornstarch. Each dietary treatment (5 total) was fed to varying numbers of rats when they reached 6 wk of age, and diets were fed for 14 d. The total number of rats used for the unsupplemented control, 48 g/kg Cys, 72 g/kg Cys, 48 g/kg Cys-Cys, or 72 g/kg Cys-Cys diets, was 32, 24, 12, 28, and 22, respectively. Time-course percentage of survival data were calculated by dividing the daily number of live rats by the total number of rats per treatment.

Pig experiments

All experimental procedures were approved by the University of Illinois Animal Care and Use Committee. Pigs resulting from the cross of PIC 337 males with PIC Camborough 22 females were obtained from the University of Illinois Swine Farm at weaning (17–21 d of age) and fed a starter diet [213 g/kg CP, 15 g/kg Lys, 3.7 g/kg Met, 4.7 g/kg cyst(e)ine] appropriate for this age pig. Pigs were group-housed in pens (1.22 m x 1.22 m) with slatted plastic flooring in an environmentally controlled building in which a constant 24-h light schedule was maintained. Food was provided via a multihole stainless steel feeder, and water was freely accessible from a nipple waterer. After a 7-d acclimation period, pigs were randomly assigned to dietary treatments from uniform blocks based on their ancestry and body weight. Food was withheld for 12 h, and initial pig weights were recorded thereafter upon the initiation of each experiment. Comparison of cyst(e)ine sources was done using L-Cys and L-Cys-Cys in both studies.

    Experiment 1. The experimental diet (Table 1), adequate in all dietary nutrients (13), was supplemented with 0, 20, or 40 g/kg of Cys or Cys-Cys at the expense of cornstarch. Supplemental levels of 20 and 40 g/kg cyst(e)ine represent an estimated 6.5- and 13-fold excess, respectively, over the cyst(e)ine requirement for maximal growth of young pigs. Dietary treatments (5 total) were fed to 4 replicate pens of 4 pigs for 14 d. After the 14-d feeding period, all pigs were switched to a standard diet [208 g/kg CP, 14 g/kg Lys, 3.5 g/kg Met, and 4.0 g/kg cyst(e)ine] appropriate for this age pig. This diet was fed for a 7-d recovery period, after which individual pig weights and pen food intakes were recorded. Weight gain, food intake, and food efficiency (i.e., gain:food ratio) were calculated per pen of 4 pigs using cumulative data from the 14-d experimental period and 7-d recovery period.

    Experiment 2. The objective was to determine whether bicarbonate could ameliorate cyst(e)ine toxicity in pigs. The experimental diet (Table 1) was supplemented with 0 or 30 g/kg Cys or Cys-Cys with or without 10 g/kg KHCO₃ at the expense of cornstarch. Diets (6 total) were fed to 3 replicate pens of 3 pigs for 7 d, after which whole blood was collected and analyzed as outlined in chick Expt. 2. Weight gain, food intake, and food efficiency (i.e., gain:food ratio) were calculated per pen of 3 pigs.

Statistical analysis

All data were subjected to ANOVA using the General Linear Model procedure of SAS (14). Data from chick experiments were analyzed using pen means with procedures appropriate for a completely randomized design. Rat data were analyzed as a completely randomized design using observations from individual rats. The pig data were subjected to procedures appropriate for a randomized complete-block design using pen means. Values are means with pooled SEM estimates. In most cases, means separation was carried out using the least significant difference multiple comparison procedure of SAS with = 0.05. Effects of excess dietary SAA on chick plasma free SAA concentrations were evaluated using single degree of freedon contrasts between the mean responses of the 2 highest dietary SAA levels vs. the unsupplemented control diet.

	Results

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
    Chick experiment 1. Weight gain, food consumption, and gain:food ratio were reduced (P < 0.05) in a dose-dependent manner after ingesting excess Met, Cys, or Cys-Cys (Table 2). Supplementation of any SAA at 10 g/kg did not depress growth performance. Ingestion of excess Met, Cys, or Cys-Cys at 40 g/kg reduced (P < 0.05) growth by 93, 59, and 20%, respectively. However, mortality occurred only in chicks consuming excess Cys, with deaths beginning after only 3 d of feeding. After 5 d, 92% (11 of 12 total) of chicks consuming 40 g/kg supplemental Cys died, and the dietary treatment containing 30 g/kg supplemental Cys was terminated on d 5 due to a 50% (6 of 12 total) chick mortality rate. Zero deaths and only a 9% reduction in weight gain resulted from ingestion of 20 g/kg supplemental Cys for the entire 9-d period.

View larger version (13K): [in this window] [in a new window]   Figure 1  Weight gain of young rats fed graded concentrations of supplemental Cys or Cys-Cys in a casein-cornstarch diet (AIN-93G). Values are means ± SEM, n = 6. Rat Expt. 1 (dietary Cys excess); 14-d feeding period, mean initial weight 101 g, pooled SEM = 0.18. Rat Expt. 2 (dietary Cys-Cys excess); 14-d feeding period, mean initial weight 121 g, pooled SEM = 0.16 (A). Rat Expt. 3 (dietary excesses of Cys or Cys-Cys); 5-d feeding period, mean initial weight 115 g, pooled SEM = 0.08 (B).

View larger version (10K): [in this window] [in a new window]   Figure 2  Time-course survival (% of total rats per treatment, 14-d feeding period) of young rats (mean initial weight 115 g) fed graded concentrations of supplemental Cys or Cys-Cys in a casein-cornstarch diet (AIN-93G). Control animals were fed the unsupplemented basal diet (nutritionally adequate). Survival is number of live rats as a percentage of total rats per treatment at any given time-point (rat Expt. 4).

	Discussion

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
Our primary objective was to characterize upper tolerance dietary concentrations of SAA fed to chicks, rats, and pigs, with a specific focus on Cys and Cys-Cys. Although the toxic nature of both Cys and Cys-Cys has been suggested (5,15,16), to our knowledge, no studies directly comparing these redox-related SAA have been conducted. Additionally, a paucity of information exists as to ameliorative treatments for cyst(e)ine toxicity. Answers to these questions are critical to our understanding of SAA nutrition, especially considering the increased availability and use of amino acid supplements in a human clinical setting.

Not surprisingly, Met was the most growth depressing of the 3 SAA evaluated in chicks, a finding reported extensively for rats in the literature (5,7,15). In our chick model, ingestion of 40 g/kg Met reduced weight gain 93%. Methionine toxicity is known to cause histopathological lesions in the small intestine, liver, kidney, and pancreas (15), and is often characterized by the resulting hemolytic anemia and subsequent splenic hemosideroses (8,16–18). Evidence suggests this tissue damage is likely due to an intermediate in the transaminative Met degradative pathway, i.e., 3-methylthiopropionate (17,18). Our attempts to ameliorate Met toxicity in chicks using 10 g/kg supplemental Gly were only partially effective (data not shown), as previously reported (18–20).

    Cyst(e)ine toxicity in young chicks. Chicks fed 40 g/kg supplemental Cys reduced weight gain by 59%. Additionally, the chlorinated form of Cys (i.e., L-Cys · HCl) was even more growth depressing than free-base Cys when provided at equimolar dose levels (data not shown). Similar conclusions on cyst(e)ine toxicity have been reported by other investigators (21–23), although a direct comparison between Cys and Cys-Cys has not been made. In our chick studies, ingestion of 40 g/kg supplemental Cys-Cys caused only a 20% reduction in weight gain. Differential utilization of these redox-related SAA suggests physiological and/or metabolic differences may exist in chicks. Indeed, intestinal absorption of Cys-Cys has been shown to be slower than that of Cys in mammalian species (24,25), although provision of either compound in crystalline form should result in efficient absorption (8). During absorption, virtually all Cys-Cys is converted to Cys within the enterocyte (26). However, it is unknown whether these normal absorptive and redox-conversion mechanisms exist in vivo at pharmacological intakes. Overall, ingestion of excess Cys-Cys up to 40 g/kg was rather innocuous to chick growth performance, particularly when compared with similar doses of either Cys or Met (Table 2).

Apart from the growth depressing effects of cyst(e)ine, we observed a high rate of mortality in chicks consuming excess Cys, but no mortality with excess Cys-Cys. Chicks began to die after 3 d of consuming diets containing supplemental Cys at 30 or 40 g/kg. Overall, 50% of chicks fed 30 g/kg Cys and 92% of chicks fed 40 g/kg Cys died after 5 d of feeding [other chick data from our laboratory indicate that 40 g/kg of supplemental Cys from N-acetyl-L-Cys results in no mortality after 9 d of feeding (8)]. However, absolutely no mortality occurred in chicks receiving 20 g/kg Cys, and growth performance was virtually unaffected at this level of supplemental Cys. To our knowledge, such acute mortality resulting from amino acid toxicity has not been reported. These data suggest there is a fine threshold in chicks, below which Cys is innocuous to growth and food intake, and above which Cys is highly lethal. Additionally, only the reduced form of cyst(e)ine is lethal to chicks up to the dose levels used here, suggesting that cyst(e)ine lethality is related to the free sulfhydryl group in this SAA. Observationally, chicks that died from excess Cys did so in an expeditious manner, with little or no warning provided of impeding death. In fact, chicks were known to perish while actively consuming food or water. In subsequent studies, supplemental Cys was reduced to 25 g/kg, and overall, we determined that the (minimal) lethal dose at which 50% mortality occurred in chicks was 25 g/kg excess Cys, representing an estimated 6.3-fold increase over the cyst(e)ine requirement for maximal growth.

To make a valid comparison between rats and chicks, we also tested the effect of 20 or 40 g/kg supplemental Cys in chicks fed a purified diet (cornstarch-casein) similar to the diet used in our rat studies. Weight gain was reduced 54 and 74% when ingesting 20 or 40 g/kg supplemental Cys, respectively, with 88% mortality observed only with the 40 g/kg Cys diet (data not shown). Additionally, we determined that intraperitoneal injections of Cys at 300 or 600 mg/kg BW neither affected growth performance nor caused chick mortality (data not shown), as previously observed in our laboratory (27). Taken together, these data suggest that the toxic Cys effects in chicks were independent of diet composition and also that bypassing the gut minimizes the pernicious effect of excess Cys.

There existed the possibility that eating behavior of chicks (i.e., constant nibbling, many small meals) was responsible for the observed Cys toxicity (28). Thus, food-deprived chicks were oral-gavaged with distilled water or 600 mg Cys (4.0 g/kg BW) in a bolus dose. On average, control chicks gained 21 g BW and consumed 56 g of diet during the 24-h postintubation period, and no mortality occurred. In stark contrast, chicks gavaged with 600 mg Cys lost 28 g BW and consumed only 1 g of diet, and 83% of these chicks died (10 of 12 total) during the 24-h observation period (data not shown). These mortalities occurred 12 h after treatment gavage, and chicks perished while in a somnolent position. Previous work from our laboratory showed that chick mortality due to high levels of I in the diet was absent when I was administered in a bolus dose (28). The Cys phenomenon was different, however, because excess Cys resulted in similar effects regardless of the administration route.

    Amelioration of Cys toxicity in young chicks. Initially, we attempted to ameliorate Cys toxicity considering it may have chelated trace metals (29), but neither supplemental Cu (as CuSO₄ · 5H₂O) nor Se (as Na₂SeO₃) affected the growth depression due to excess Cys (data not shown). We then hypothesized that Cys may be exerting its pernicious effect through its potential as a strong reducing agent. In this regard, Cys-Cys was not lethal to chicks so it seemed logical that the free sulfhydryl group was involved in the toxic mechanism. Indeed, dietary inclusion of 7.5 or 10.0 g/kg KHCO₃ restored chick growth performance relative to the unsupplemented control diet. However, whereas chick mortality rate was reduced, it remained at 25%. Amelioration of Cys toxicity via dietary bicarbonate, however, seems counterintuitive to the argument that Cys is a bicarbonate-sensitive endogenous excitotoxin (30,31), although the bicarbonate effect we observed may have been independent of the proposed Cys neurotoxicity mechanism. Moreover, we observed no significant effect of bicarbonate treatment on blood pH, bicarbonate, or base excess in chicks consuming excess Cys (data not shown).

Further attempts to ameliorate Cys toxicity led to the idea that it might be possible to counteract its strong reducing agent activity by including a strong oxidizing agent (i.e., H₂O₂) in the drinking water. An initial study confirmed that chicks were highly sensitive to the ingestion of H₂O₂ and could tolerate only a low dose (0.05% final concentration; data not shown). Inclusion of this concentration of H₂O₂ in the drinking water completely abolished chick deaths due to ingestion of excess Cys, but it had only a minimal effect in restoring growth performance. Finally, we evaluated the well-known principle that increased dietary CP concentration may minimize the noxious effects of consuming a single excess amino acid (5,23). Supplemental dietary Cys at 25 g/kg depressed weight gain in chicks fed corn-soybean meal diets containing 180 g/kg or 240 g/kg CP, but weight gain of chicks fed 300 g/kg CP was not affected by this level of excess Cys (data not shown).

    Cyst(e)ine toxicity in young rats. Not surprisingly, the bulk of SAA toxicity research has been done in rats (5,15,16,23). A summary of previous studies suggests reduced weight gain and food intake result when rats are fed low-protein purified diets containing 5.0–100 g/kg supplemental Cys or Cys-Cys (15). However, a paucity of information exists comparing the relative toxic effects of Cys and Cys-Cys. Our results showed that Cys-Cys might be slightly less growth depressing than Cys at graded supplemental doses up to 24 g/kg, but both Cys and Cys-Cys caused a 74% reduction in weight gain at 48 g/kg. This growth depression was accompanied by a 39% reduction in food intake (data not shown). Additionally, when directly comparing Cys and Cys-Cys in an acute feeding study, we showed that either redox form caused acute body weight loss. Therefore, as previously suggested (5), a period of adjustment to excess cyst(e)ine intake must occur in feeding studies of longer duration.

Rat mortality due to excess cyst(e)ine has been reported by other investigators (5,21), with Cys generally reported to cause more deaths than Cys-Cys. Our results provide evidence to the contrary. Although both forms of cyst(e)ine were equally growth depressive, Cys-Cys was more pernicious than Cys in terms of lethality at 72 g/kg of supplemental intake. Explanation for this discrepancy is unclear, but careful attention should be given to the age of the animals, composition of the basal diet, previous diet provisions, and length of feeding periods among individual experiments.

    Cyst(e)ine toxicity in young pigs. To our knowledge, no research has been conducted on cyst(e)ine toxicity in pigs. Because this species serves as an excellent model for humans, this research may be useful for defining tolerable upper limits for cyst(e)ine as promoted by ICAAS. We used 2 doses each of Cys and Cys-Cys; one we expected to be relatively innocuous and the other was found to be lethal in chicks. After ingestion for 14 d, pigs exhibited severely depressed weight gain, food intake, and gain:food ratio, regardless of the cyst(e)ine source. Overall, excess cyst(e)ine at 40 g/kg reduced weight gain by 84%. Previous investigations of amino acid excess in young pigs clearly showed that Met was the most growth depressing (among all indispensable amino acids) and reduced weight gain up to 54% when administered at 40 g/kg over a 16-d feeding period (6,32). Our results suggest that excess dietary cyst(e)ine is even more noxious to young pigs than Met in terms of reduced growth performance.

No pig mortality resulted from feeding 40 g/kg excess Cys, a dose level that is absolutely lethal for chicks. A number of possible explanations may account for this species difference: 1) the excess cyst(e)ine level was below the pig's lethal threshold; 2) absorption of crystalline cyst(e)ine is incomplete in pigs at excessive levels; and 3) first-pass intestinal metabolism of excess cyst(e)ine is sufficient to circumvent lethal effects (e.g., the excretory capacity via the urine is adequate for detoxification). Further pig studies are needed to define an upper tolerance limit for cyst(e)ine and thereby provide evidence for a dose that causes mortality. It is generally accepted that crystalline amino acids are completely absorbed (8,33), although these conclusions are based on studies done with amino acid levels below, rather than above, minimal requirement concentrations. Finally, evidence in support of extensive intestinal Cys metabolism has been suggested (34–37). Therefore, due to the vast intestinal surface area and tissue mass in pigs (relative to chicks and rats), it is possible that pigs are able to avoid the lethal effects of cyst(e)ine. The growth depressing effect of cyst(e)ine is undeniable, but it clearly raises the question of whether excess cyst(e)ine may have merely been serving as an anorectic agent in pigs (38). Unlike in chicks, KHCO₃ was unable to ameliorate the growth depressive effects of excess cyst(e)ine in pigs. Further studies are warranted to determine the mechanistic route behind the noxious nature of excess dietary Cys and Cys-Cys.

    Histopathological/hematological findings. Studies attempting to elucidate the toxic mechanism of Cys in chicks are ongoing in our laboratory. Initial histopathological examination of all organ systems revealed no gross lesions or abnormal findings compared with control and Cys affected chicks. Heart and brain tissues were further examined, but no noteworthy lesions were found. Additionally, hematoxylin basic fuchsin picric acid staining showed that chick deaths were not due to myocardial infarction. Verhoeff's Van Gieson staining suggested no difference in the architecture of the elastin fibers in the great vessels of chicks that died from ingesting excess Cys. Future chick studies will focus on the proposed mechanistic action of Cys as an endogenous excitotoxin (39). In this regard, Cys has been shown to selectively activate N-methyl-D-aspartate receptors and cause brain damage similar to Glu (31,40). Finally, the chemical nature of Cys (i.e., the free sulfhydryl moiety) suggests Cys may cause toxicity through autoxidation, resulting in the generation of H₂O₂ in vivo (41).

After ingesting excess dietary SAA for 9 d, we expected plasma free SAA concentrations to be dramatically altered in chicks. Although plasma free Met was markedly increased due to excess dietary Met, plasma Cys concentrations did not change by ingestion of excess dieary Met, Cys, or Cys-Cys. Though not shown, the mean plasma free Cys concentrations from chicks receiving 30 or 40 g/kg for 5 d was 458.1 µmol/L. Thus, after 5 d of ingesting excess levels of Cys (i.e., levels that ultimately resulted in chick mortality), plasma free Cys was not affected. Regulation of hepatic Cys catabolism, including increased Cys dioxygenase activity and reduced cysteinesulfinate decarboxylase activity, occurs in the presence of excess dietary SAA (35,42,43). Although metabolic adaptation to excess dietary cyst(e)ine is likely, these results are still striking because food was not withheld from chicks prior to blood collection.

Overall, the plasma free SAA data provide little mechanistic evidence to explain the toxic nature of excess dietary Cys in chicks. Although we cannot exclude the possibility that the observed Cys toxicity was due to the buildup of Cys catabolites (e.g., NH₃ or H₂S), the effect of excess dietary Cys on blood pH was minimal. It should be noted, however, that the sudden deaths in chicks fed excess Cys are somewhat consistent with acute hyperammonemia (44,45). Elucidating a direct link between excess dietary Cys and ammonia toxicity obviously warrants further investigation, but differences in nitrogen excretion exist between the 3 species we studied (i.e., chicks are uricotelic, whereas rats and pigs are ureotelic).

Preliminary evidence of Cys-Cys toxicity in rats included the occurrence of pulmonary congestion, pleural effusion, and red spots on the lungs (data not shown). Additionally, hepatic perilobular necrosis was induced by excess Cys-Cys. Among biomarkers evaluated, alkaline phosphatase and creatine phosphokinase were the most sensitive [i.e., elevated at the lowest dietary cyst(e)ine concentration] in young rats. Similar findings could not be verified in young pigs [i.e., neither excess Cys nor Cys-Cys affected alkaline phosphatase, aspartate aminotransferase, gamma-glutamyl transpeptidase, or creatine phosphokinase after the 14-d feeding period (data not shown)]. Additionally, necropsy of pigs previously fed 40 g/kg Cys or Cys-Cys for 14 d showed no histopathological lesions of any kind.

Cyst(e)ine is sold in various countries as an over-the-counter pharmaceutical as well as a dietary supplement. When sold as an over-the-counter pharmaceutical, cyst(e)ine dosage levels are regulated by governing bodies specific to each country. Recommended daily intakes of pharmaceutical cyst(e)ine are required on the product label with suggested intakes generally <1 g/d. However, such restrictions do not exist for cyst(e)ine products used as dietary supplements. Hence, a recent objective of ICAAS has been to define upper tolerance limits for commercially available amino acids, including SAA. Without regulatory control of these over-the-counter nonprescription cyst(e)ine products, careful attention must be given to their efficacy and toxicological effects.

Results from the cyst(e)ine toxicity studies herein support the need for more studies of upper tolerance levels of SAA. Amino acid supplements are normally consumed by humans as bolus pills, and usage instructions are often ambiguous. The growth depressing dose levels of excess Cys and Cys-Cys used in this study were higher (relative to the minimal requirement) than those consumed as dietary supplements by humans. Nonetheless, we have shown that both Cys and Cys-Cys can be severely growth depressing in 3 different animal models. Moreover, lethality of high dose levels was exhibited for Cys in chicks and for Cys-Cys in rats. For these reasons, upper tolerance limits for Cys and Cys-Cys need to be defined for humans. Because daily dose levels for over-the-counter pharmaceutical cyst(e)ine have generally been <1 g/d, a similar dietary intake level for cyst(e)ine supplements would seem prudent.

	FOOTNOTES

 
³ Abbreviations used: BW, body weight; CP, crude protein; SAA, sulfur amino acid.

Manuscript received 21 September 2006. Initial review completed 31 October 2006. Revision accepted 17 November 2006.

	LITERATURE CITED

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 

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