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© 2008 American Society for Nutrition

Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions

Excess Dietary L-Cysteine Causes Lethal Metabolic Acidosis in Chicks1

Ryan N. Dilger* and David H. Baker

Department of Animal Sciences and Division of Nutritional Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801

* To whom correspondence should be addressed. E-mail: rdilger2{at}illinois.edu.


   ABSTRACT
TOP
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
A 72-h time-course study was conducted to elucidate the physiological mechanism underlying cysteine (Cys) toxicity in chicks beginning at 8-d posthatch. Biochemical markers quantified in plasma and liver samples collected from chicks receiving 30 g/kg excess dietary Cys were compared with baseline measurements from chicks receiving an unsupplemented corn-soybean meal diet over a 72-h feeding period. Concomitant with chick mortality were indices of acute metabolic acidosis, including a rapid increase (P < 0.001) in anion gap that resulted from a reduction (P < 0.001) in plasma HCO3– of ~40% and a 2.8-fold increase (P < 0.001) in plasma sulfate in chicks receiving excess Cys. Additionally, provision of 30 g/kg excess Cys resulted in a 1.5-fold increase (P < 0.05) in hepatic oxidized glutathione compared with the 0-h control time-point. Excess dietary Cys did not affect plasma free Met, but plasma free Cys increased (P < 0.05) from 89 to 107 µmol/L at 12 h and remained elevated through 36 h. Strikingly, ingestion of 30 g/kg excess Cys caused more than a doubling (P < 0.001) of plasma free cystine, the oxidized form of Cys, beginning 12 h after initiating the study, and it remained elevated throughout the 72-h feeding period. Taken together, these data suggest that ingestion of 30 g/kg excess L-Cys causes both acute metabolic acidosis and oxidative stress in young chicks when fed a nutritionally adequate, corn-soybean meal diet.


   Introduction
TOP
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Both the reduced (Cys) and oxidized (Cys-Cys) forms of cyst(e)ine support animal growth equally well when provided in a cyst(e)ine-deficient and Met-adequate diet (1). However, we previously showed that ingestion of 25 g/kg or more excess Cys, but not Cys-Cys or N-acetyl-L-Cys, was acutely lethal for chicks over a 9-d feeding period (2). Indirect evidence from our previous work suggested that some of the toxic effects of excess Cys could be explained by oxidative stress due to the strong reducing nature of Cys. Thus, ingestion of excess Cys may be expected to affect the overall redox status of the chick (3,4). Importantly, the close relationship of sulfur amino acids (SAA)2 and antioxidant status [e.g. glutathione (GSH)] has the potential to increase the popularity of sulfur-containing compounds as nonprescription over-the-counter pharmaceuticals and their use in human clinical medicine. Thus, characterizing the link between dietary SAA intake and redox status is important, especially considering that oxidative stress has been implicated as part of the normal aging process (4).

Metabolism of Cys provides essential substrates (e.g. GSH, taurine, and inorganic sulfur) for synthetic reactions, detoxification and antioxidative processes, osmotic regulation, and nervous system function (5). However, regulation of endogenous Cys concentrations is crucial due to the potential for this SAA to cause tissue damage, especially in the brain and retina (6–8). Whereas many studies have used rodent models to investigate the excitotoxic effects of Cys on the immature brain, our previously reported species comparison (2) revealed that excess L-Cys ingestion is lethal for chicks but not for pigs or rats. Oxidation of Met or Cys to inorganic sulfate results in the production of 2 protons, and consumption of diets containing excess protein or SAA has been shown to cause acidosis in both rats and humans (9–12). Additionally, both cysteinesulfinate-dependent and -independent pathways culminate in synthesis of inorganic sulfate (13) and although sulfate is the 4th most abundant anion in plasma, little is known about factors regulating its homeostasis in animals (14). This is potentially important, because sulfate levels must be tightly controlled to maintain proper acid-base balance. Under normal circumstances, renal clearance of sulfate can accommodate excess sulfate loads (15), but 7- to 24-fold increases in plasma sulfate are common in humans experiencing renal failure (16–19).

The interrelated nature of Cys metabolism with antioxidant status and acid-base balance makes this SAA especially intriguing. Thus, it is important to elucidate the biological adaptation of animals to ingestion of excess Cys, and the chick serves as a unique model in this respect due to the systemic and lethal nature of Cys toxicity in this species. We hypothesized that indices of both redox status and acid-base balance would respond to ingestion of excess Cys. The chick study described herein was designed to evaluate the physiological adaptation of chicks upon ingesting 30 g/kg excess Cys over a 72-h feeding period.


   Materials and Methods
TOP
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 LITERATURE CITED
 
All experimental procedures were approved by the University of Illinois Animal Care and Use Committee. Male chicks (New Hampshire male x Columbian female) obtained from the University of Illinois Poultry Farm were used to investigate the mechanism underlying acute lethality due to ingestion of excess L-Cys. 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 typical corn-soybean meal starter diet (i.e. pretest diet; Table 1) that provided 230 g/kg crude protein (CP) and was adequate in all dietary nutrients (20). Without an overnight food deprivation period, chicks were weighed, wing-banded, and randomized to dietary treatments on d 8, so that average initial pen weights and weight distributions were similar across treatments. Chicks were housed 4/pen and individual chicks were removed from independent pens at appropriate time points during the 72-h study.


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  		TABLE 1 Composition of pretest and experimental basal diets

 
The experimental corn-soybean meal basal diet (Table 1) was formulated to be nutritionally adequate (20) for this age chick and it was similar to the pretest diet in both ingredient composition and nutrient profile. Diets and tap water were freely available to chicks at all times. The basal diet was supplemented with or without 30 g/kg excess L-Cys (at the expense of cornstarch) to produce the experimental diets, and chicks received these diets for 72 h beginning on d 8 posthatch.

    Sample collection. At the appropriate time point, 5 chicks per treatment were randomly selected from independent pens, killed via CO2 asphyxiation, and blood was collected via cardiac puncture into heparinized evacuated vials. Liver samples were collected and immediately snap-frozen in liquid nitrogen pending analysis. Prior to receiving experimental diets, samples were collected from 5 chicks to provide common reference measurements (0-h time point); these control chicks were fed the nutritionally adequate pretest diet (similar in composition to the experimental diet) and importantly, these measurements were quantified on non-food–deprived chicks. Beyond the 0-h time point, samples from 5 chicks receiving the 30 g/kg excess Cys diet were harvested every 12 h throughout the 72-h feeding period. Additionally, samples from 5 chicks receiving the unsupplemented basal diet were harvested at 36 and 72 h to provide baseline measurements with respect to time.

    Analytical procedures. Plasma samples collected from non-food–deprived chicks were quantified for biochemical markers by spectrophotometric analysis. Blood biochemistry was assayed on the Hitachi 917 analyzer (Roche) using Roche diagnostic reagents. The anion gap was defined as the difference between the concentrations (mmol/L) of unmeasured anions and cations and was calculated as follows: (Na+ + K+) – (Cl– + HCO3–) (21).

Simultaneous chromatographic separation of plasma free SAA (Cys, Cys-Cys, and Met) was performed using an HPLC procedure previously described (22) with the following modifications. Plasma samples, deproteinized with an equal volume of 3.5% sulfosalicylic acid (23), were analyzed using a DX-500 liquid chromatograph (Dionex) fitted with a Dionex OmniPac PCX-500 column (4 x 250 mm) and protected by 2 similar guard columns (4 x 50 mm). A 50-µL volume of the mobile phase (0.15 mol/L sodium perchlorate, 5% acetonitrile, 20 mmol/L perchloric acid) was injected at a flow rate of 1.2 mL/min over a 50-min collection period. This cation exchange method employed integrated pulsed amperometric detection using an Au working electrode.

Plasma samples, deproteinized with an equal volume of 3.5% sulfosalicylic acid (23), were analyzed using a DX-600 liquid chromatograph (Dionex) for determination of inorganic sulfate. The system was fitted with a Dionex IonPak AS11 column (4 x 250 mm), Dionex AG11 guard (4 x 50 mm), and Dionex GS50 gradient pump. A programmed gradient of KOH (ranging from 2–30 mmol/L) was injected at a flow rate of 2.0 mL/min over a 15-min collection period. Ion suppression of the column eluent was accomplished using the Dionex ASRS Ultra II 4-mm suppressor operating in the autosuppression recycle mode. Detection was accomplished using the Dionex ED50 electrochemical detector fitted with a conductivity detector.

Liver tissue (0.6–1.1 g) was homogenized in ice-cold 100 mmol/L potassium phosphate buffer, pH 7.4 (10% wt:v), and immediately centrifuged (47,000 x g; 10 min; 4°C). To reduce the incidence of subsequent lipid peroxidation, butylated hydroxytoluene (3 µL) was added to a homogenate aliquot for malondialdehyde (MDA) determination. A separate aliquot was immediately deproteinized using 5% meta-phosphoric acid (wt:v) at a 2:1 acid:homogenate ratio and centrifuged at 1,000 x g; 5 min to stabilize samples for GSH determination. Protein concentration of liver homogenates was determined by the Bradford method (24) using samples that had been stored at 4°C.

Hepatic reduced GSH was quantified using a commercial assay kit (Northwest Life Science Specialties) based on the original method described by Tietze (25). Briefly, total GSH was quantified following enzymatic recycling with GSH reductase and kinetic spectrophotometric analysis at 408 nm. Oxidized GSH was quantified similarly in the presence of 4-vinylpyridine, which nonspecifically scavenges free thiol moieties. Reduced GSH was calculated as the difference between total and oxidized GSH.

We determined susceptibility of liver tissue to lipid peroxidation by quantifying MDA accumulation using a commercial assay kit (Northwest Life Science Specialties). Because the presence of hemoglobin interferes with the analysis of MDA, liver homogenates were first subjected to butanol extraction followed by back-extraction with NaOH. Adducts of MDA and thiobarbituric acid were quantified in an acidic medium using 3rd derivative results from a spectral analysis (400–700 nm), which allows compensation for the nonlinear baseline typical of biological samples.

    Statistical analysis. All data were subjected to ANOVA using the GLM procedure of SAS (SAS Institute). Data were analyzed using individual chicks as the experimental unit, with procedures appropriate for a completely randomized design. Data are presented as mean values with pooled SEM from 5 independent chicks at each time point during the 72-h feeding period. Data collected from time points after 0 h were compared with control data (i.e. 0-h control) using a Dunnett's multiple comparison test assuming an {alpha} level of 0.05. Nonorthogonal contrasts comparing the effects of 30 g/kg excess Cys to baseline data were tested at 36 and 72 h.


   Results
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 LITERATURE CITED
 
Provision of 30 g/kg excess dietary Cys decreased (P < 0.05) chick body weight gain by 11% and feed intake by 15% (data not shown). Non-food–deprived chicks harvested prior to receiving the experimental diets had normal clinical chemistry values for chickens (Table 2). Biomarkers not affected (data not shown) by excess dietary Cys at either 36 or 72 h included glucose, total bilirubin, cholesterol, the albumin:globulin ratio, and blood enzymes (alkaline phosphatase, aspartate aminotransferase, creatine kinase, {gamma}-glutamyltransferase, and sorbitol dehydrogenase).


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  		TABLE 2 Biomarkers of acid-base balance and oxidative stress in chicks during a 72-h feeding period1

 
Chicks remained physiologically stable over the 72-h feeding period as shown by the lack of differences from control values obtained from chicks fed the control diet for all plasma biomarkers except Ca2+. Concentrations of all other biomarkers did not differ (P > 0.05) from control values at either 36 or 72 h, and linear responses (i.e. with a positive or negative slope) over the 72-h feeding period were observed only for Mg2+, K+, and ammonia (P < 0.10; data not shown). Whereas ingestion of the unsupplemented basal diet increased plasma K+ and decreased plasma Cl– over time, Cys supplementation caused K+ to be lower (P = 0.04) and Cl– to be higher (P < 0.001) at 72 h.

Just 12 h after initiating the study, plasma HCO3– was reduced by 32% (P = 0.001) in chicks receiving 30 g/kg excess Cys (Fig. 1A). Overall, ingestion of excess Cys reduced HCO3– from 26.1 mmol/L (baseline) to 15.4 mmol/L (mean of all time points where excess Cys was ingested). Moreover, because Na+, K+, and Cl– were not greatly affected by Cys supplementation, the anion gap was increased (P < 0.01) at 12, 24, 36, and 72 h relative to the 0-h control value (Fig. 1B). Plasma sulfate concentrations from chicks fed 30 g/kg excess Cys were elevated 3.4- and 2.3-fold (P < 0.01) at 36 and 72 h, respectively, after initiating the study (Fig. 1C). Sulfate concentrations of chicks fed the unsupplemented basal diet remained stable over the 72-h feeding period. Plasma HCO3– concentrations decreased (P < 0.001) and anion gap and sulfate concentrations increased (P < 0.001) in chicks receiving excess Cys at 36 and 72 h compared with baseline estimates from chicks receiving the unsupplemented basal diet.


Figure 1
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  		FIGURE 1  Plasma concentrations of HCO3– (A), anion gap (B), and sulfate (C) over a 72-h feeding period. Anion gap was calculated as (Na+ + K+) – (Cl– + HCO3–) with all ions expressed as mmol/L. Control values (0-h time point) were taken from chicks that had been fed the pretest diet (i.e. prior to receiving the experimental basal diet). Beginning on d 8 posthatch, chicks received the corn-soybean meal basal diet (Table 1) supplemented without (basal, B) or with 30 g/kg excess L-Cys. At 36 and 72 h, plasma HCO3–, anion gap, and plasma sulfate in chicks fed excess Cys differed (P < 0.05) from those fed the unsupplemented basal diet. Values are means ± SEM of 5 independent chicks per time point. *Different from control, P < 0.05. Pooled SEM for plasma HCO3–, anion gap, and sulfate were 1.7, 1.1, and 0.09 mmol/L, respectively.

 
Hepatic reduced GSH did not remain stable over the 72-h period; this biomarker decreased linearly (P < 0.05) in chicks fed the unsupplemented basal diet (Table 2), and 30 g/kg excess dietary Cys did not affect this time-dependent decrease. In contrast, oxidized GSH was stable over time, and ingestion of excess Cys caused increases (P < 0.05) of up to 1.5-fold at 36 and 72 h. Additionally, the ratio of reduced:oxidized GSH in chicks fed either the unsupplemented basal diet or the basal diet supplemented with 30 g/kg excess Cys was lower (P < 0.05) at 36 h. At 72 h, the ratio of reduced:oxidized GSH did not differ from 0-h control values for chicks receiving the unsupplemented basal diet, but the ratio remained low for chicks ingesting excess Cys (P < 0.05). Oxidative stress could not be confirmed by measurement of hepatic lipid peroxidation (i.e. MDA).

Plasma free Met was unaffected by ingestion of excess dietary Cys (Fig. 2A). Baseline plasma Met concentrations remained stable over time, and unsupplemented and Cys-supplemented diets did not differ at either 36 or 72 h. However, ingestion of 30 g/kg excess dietary Cys increased (P = 0.001) plasma free Cys 20% by 12 h and this biomarker remained elevated (P < 0.05) above baseline values at 24 and 36 h (Fig. 2B). After the 36 h time point, excess dietary Cys caused plasma free Cys to be numerically lower than baseline values, and at 72 h ingestion of 30 g/kg Cys tended to cause a lower (P = 0.08) plasma Cys concentration relative to the unsupplemented basal diet.


Figure 2
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  		FIGURE 2  Plasma concentrations of Met (A), Cys (B), and Cys-Cys (C) over a 72-h feeding period. Control values (0-h time point) were taken from chicks that had been fed the pretest diet (i.e. prior to receiving the experimental basal diet). Beginning on d 8 posthatch, chicks received the corn-soybean meal basal diet (Table 1) supplemented without (basal, B) or with 30 g/kg excess L-Cys. At 36 and 72 h, plasma Cys and Cys-Cys in chicks fed excess Cys differed (P < 0.05) from those fed the unsupplemented basal diet. Values are means ± SEM of 5 independent chicks per time point. *Different from control, P < 0.05. Pooled SEM for plasma Met, Cys, and Cys-Cys were 8.8, 5.4, and 22.4 µmol/L, respectively.

 
Provision of excess dietary Cys increased (P = 0.001) plasma free Cys-Cys from 95 to 202 umol/L just 12 h after initiating the study (Fig. 2C). Plasma Cys-Cys remained elevated (P < 0.05) above the 0-h control value at all time points due to ingestion of 30 g/kg excess Cys, and plasma Cys-Cys values were twice as high (P < 0.05) as baseline values at 36 and 72 h. The ratio of oxidized:reduced Cys (i.e. Cys-Cys:Cys) increased from ~1.0 for chicks fed the unsupplemented basal diet to 2.7 for chicks receiving excess dietary Cys at 48, 60, and 72 h. Overall, consumption of the unsupplemented basal diet did not affect time-course measurements of either plasma free Cys or Cys-Cys.


   Discussion
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
LITERATURE CITED
 
Recent evidence from our laboratory suggested that L-Cys, but not L-Cys-Cys, was lethal when ingested by young chicks at 25 g/kg or greater in excess of the dietary Cys requirement (2). The time-course study reported herein sought to characterize the physiological mechanism underlying this phenomenon. We theorized that disturbances in the acid-base balance or oxidative stress may play a role in the acute Cys toxicity in chicks. Our previous results suggested that the dietary addition of 10 g/kg KHCO3 or supplementation of drinking water with 0.05% H2O2 could ameliorate the growth depression and mortality, respectively, associated with ingesting 25 g/kg excess Cys (2). Thus, we previously showed evidence that both acid-base balance and oxidative stress were implicated in Cys toxicity; the results reported herein lend further credence to this theory.

The anion gap concept is based on the theory of electroneutrality, where the anion gap represents the difference between concentrations of unmeasured anions and cations (21). We observed a significant decrease in HCO3– and an increase in anion gap just 12 h after initiating the chick study. Interestingly, total protein, albumin, and inorganic P remained stable over time in chicks consuming excess Cys; this is an important point, because these 3 biomarkers constitute well over two-thirds of the anion gap estimate (21). Thus, the dramatic increase in anion gap we observed suggested the presence of an unmeasured anion. Inorganic sulfate is an often-cited example of such an anion (26) and production of sulfate is an intuitive outcome in this situation, because chicks ingested excess Cys 6-fold above the dietary requirement.

Plasma sulfate concentrations in chicks receiving excess Cys were elevated 3.4- and 2.3-fold at 36 and 72 h, respectively, compared with chicks receiving the unsupplemented basal diet. We believe that acute accumulation of inorganic sulfate, a strong anion, was sufficient to cause lethal metabolic acidosis in the chick. Incidentally, consumption of pure inorganic sulfur in the form of "flowers of sulfur," a fine yellow powder that is >99.5% sulfur, has been shown to induce acute metabolic acidosis in humans (16,27). Previous studies also suggested that provision of excess SAA or protein leads to metabolic acidosis in both rats and humans (9–12), but associated lethality has not been reported in other species. Therefore, it is logical to conclude that the chick either handles excesses of SAA differently or is simply less tolerant of disturbances in acid-base balance compared with mammalian species. Using a rat model, Bella and Stipanuk (9) suggested excess SAA intake increased partitioning to taurine vs. sulfate, which may be interpreted as an attempt to preclude acidosis by shunting sulfur to an innocuous product instead of sulfate. If a similar response occurred in the chick, this adaptation was evidently insufficient to preclude acidosis. Thus, we speculate that either the chick could not maintain sufficient renal clearance of sulfate or that regulatory control over Cys catabolism is poor in this species. Regardless, the chick appears to be a sensitive model for both Cys toxicity and metabolic acidosis.

There is evidence to suggest that thiosulfate, another Cys catabolite, impairs sulfate transporters and overall renal sulfate excretion in chickens (28,29). Undoubtedly, impaired sulfate excretion along with little change in plasma Cl– concentration (i.e. normochloremia) would increase anion gap, and increased plasma sulfate would be expected if renal sulfate excretion was compromised. In our chick study, we attempted to quantify thiosulfate, but degradation of this transient compound to sulfate is thought to occur at a rate of 5–10%/d, which precluded accurate analysis. Under normal circumstances, renal clearance of sulfate can accommodate excess sulfate loads (15), but 7- to 24-fold increases in plasma sulfate are common in human subjects experiencing renal failure (16–19). Therefore, a quantitative measure of plasma sulfate provides crucial evidence to confirm that the presence of sulfate per se is directly responsible for high anion gap metabolic acidosis in our chick model. Additionally, the decrease in plasma HCO3– concentration was similar in magnitude to the increase in anion gap, lending credence to the likelihood that an unmeasured anion (e.g. sulfate) was being produced faster than it could be excreted by the avian kidney (26).

Whole-body Cys concentrations are tightly regulated by the liver due to potential toxicity of this SAA (5,30). Our chick study was designed to characterize adaptive physiological responses to excess dietary Cys and we showed that chicks quickly responded by eliminating the reduced form of Cys. However, we did not expect the more than doubling of plasma free Cys-Cys that was sustained throughout the 72-h study. Ingestion of excess Cys caused a dramatic shift in plasma redox status; the ratio of oxidized:reduced Cys (i.e. Cys-Cys:Cys ratio) increased from ~1.0 for chicks consuming the unsupplemented basal diet to a maximum of 2.7 for chicks ingesting 30 g/kg excess Cys. Because cyst(e)ine constitutes the major extracellular low-molecular-weight thiol in the body thiol pool (3), it appears that chicks compensate for ingestion of excess Cys by quickly shifting the redox status toward an oxidative state. Similar conclusions may be drawn from chick liver data suggesting that greater concentrations of oxidized GSH were present 36 and 72 h after initiating the study. Moreover, the ratio of reduced:oxidized GSH, a sensitive marker of oxidative stress (31), was shown to return to normal by 72 h for chicks receiving the unsupplemented basal diet, but chicks ingesting excess Cys maintained a depressed ratio of ~1600 throughout the study. This is an indication that Cys may be serving a prooxidant role in this paradigm, even though Cys availability is normally limiting for GSH production. However, hepatic MDA, a marker of lipid peroxidation, showed no appreciable or consistent response to ingestion of excess Cys. The link between such shifts in redox status and animal health is complicated, but the implications of oxidative stress are becoming better characterized. As such, it is now clear that oxidative stress and aging are closely related (4). Thus, characterizing physiological responses to dietary insults and the effects on whole-body redox status are certainly important.

Our time-course study provides a biochemical explanation for the observed lethality of excess dietary Cys, but it falls short of explaining why neither Cys-Cys nor N-acetyl-L-Cys are lethal to chicks when provided in the pharmacologic dosing range (2,32). Provision of excess Cys-Cys is innocuous for chicks when provided in excess at levels isosulfurous to lethal levels of Cys, even though β-cleavage of Cys-Cys by cystathionine {gamma}-lyase should result in sulfate formation (13). Moreover, the lack of toxicity when feeding N-acetyl-L-Cys suggests that either the free thiol moiety is not directly involved or that small differences in metabolism of N-acetyl-L-Cys are sufficient to preclude toxicity. Elucidating the differential metabolisms of Cys, Cys-Cys, and N-acetyl-L-Cys may also prove useful in understanding the role of redox status in health. It appears that the use of sulfur-containing compounds is becoming ever more popular in clinical medicine and as nonprescription over-the-counter pharmaceuticals, which may be due to the relationship of SAA and antioxidant status.

From our time-course feeding study, we conclude that ingestion of 30 g/kg excess Cys (6-fold above the dietary Cys requirement) causes acute lethality in chicks, primarily due to metabolic acidosis. This lethal acidosis manifested as a significant increase in plasma sulfate, which occurred concomitantly with an acute reduction in plasma HCO3–. Moreover, chicks rapidly adapted to excess dietary Cys as evidenced by a rapid depression in plasma Cys and a sustained elevation in plasma Cys-Cys. Ultimately, and perhaps secondary to metabolic acidosis, excess dietary Cys caused increased oxidative stress as evidenced by shifts in the redox status of plasma free SAA and hepatic GSH. However, even with rapid changes in the acid-base balance and redox status, chicks were unable to fully resist acute systemic effects due to ingestion of excess Cys. The acute and rapid lethality in chicks but not in rats or pigs fed a 2.5 g/kg or higher dietary concentration of supplemental Cys (2) is striking and we have not resolved that issue here. One cannot help but wonder if uric acid rather than urea production in the avian is somehow involved, particularly because uric acid, like Cys, has reducing agent activity. This point may be of particular interest to human health because of the potential to impact disease states associated with accumulation of uric acid (e.g. gout).


   FOOTNOTES
 
1 Author disclosures: R. N. Dilger and D. H. Baker, no conflicts of interest. Back

2 Abbreviations used: CP, crude protein; GSH, glutathione; MDA, malondialdehyde; SAA, sulfur amino acid. Back

Manuscript received 23 April 2008. Initial review completed 6 June 2008. Revision accepted 20 June 2008.


   LITERATURE CITED
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 

1. Baker DH. Comparative species utilization and toxicity of sulfur amino acids. J Nutr. 2006;136:S1670–5.[Abstract/Free Full Text]

2. Dilger RN, Toue S, Kimura T, Sakai R, Baker DH. Excess dietary L-cysteine, but not L-cystine, is lethal for chicks but not for rats or pigs. J Nutr. 2007;137:331–8.[Abstract/Free Full Text]

3. Moriarty-Craige SE, Jones DP. Extracellular thiols and thiol/disulfide redox in metabolism. Annu Rev Nutr. 2004;24:481–509.[CrossRef][Medline]

4. Jones DP. Extracellular redox state: refining the definition of oxidative stress in aging. Rejuvenation Res. 2006;9:169–81.[CrossRef][Medline]

5. Stipanuk MH, Londono M, Lee JI, Hu M, Yu AF. Enzymes and metabolites of cysteine metabolism in nonhepatic tissues of rats show little response to changes in dietary protein or sulfur amino acid levels. J Nutr. 2002;132:3369–78.[Abstract/Free Full Text]

6. Karlsen RL, Grofova I, Malthe-Sorenssen D, Fonnum F. Morphological changes in rat brain induced by L-cysteine injection in newborn animals. Brain Res. 1981;208:167–80.[CrossRef][Medline]

7. Pedersen OO, Karlsen RL. The toxic effect of L-cysteine on the rat retina. A morphological and biochemical study. Invest Ophthalmol Vis Sci. 1980;19:886–92.[Free Full Text]

8. Mathisen GA, Fonnum F, Paulsen RE. Contributing mechanisms for cysteine excitotoxicity in cultured cerebellar granule cells. Neurochem Res. 1996;21:293–8.[Medline]

9. Bella DL, Stipanuk MH. Effects of protein, methionine, or chloride on acid-base balance and on cysteine catabolism. Am J Physiol. 1995;269:E910–7.[Medline]

10. Trilok G, Draper HH. Sources of protein-induced endogenous acid production and excretion by human adults. Calcif Tissue Int. 1989;44:335–8.[Medline]

11. Trilok G, Draper HH. Effect of a high protein intake on acid-base balance in adult rats. Calcif Tissue Int. 1989;44:339–42.[Medline]

12. Lemann J Jr, Relman AS. The relation of sulfur metabolism to acid-base balance and electrolyte excretion: the effects of DL-methionine in normal man. J Clin Invest. 1959;38:2215–23.[Medline]

13. Stipanuk MH. Sulfur amino acid metabolism: pathways for production and removal of homocysteine and cysteine. Annu Rev Nutr. 2004;24:539–77.[CrossRef][Medline]

14. Fernandes I, Hampson G, Cahours X, Morin P, Coureau C, Couette S, Prie D, Biber J, Murer H, et al. Abnormal sulfate metabolism in vitamin D-deficient rats. J Clin Invest. 1997;100:2196–203.[Medline]

15. Lotspeich WD. Renal tubular reabsorption of inorganic sulfate in the normal dog. Am J Physiol. 1947;151:311–8.[Free Full Text]

16. Blum JE, Coe FL. Metabolic acidosis after sulfur ingestion. N Engl J Med. 1977;297:869–70.[Medline]

17. Cole DE, Evrovski J. The clinical chemistry of inorganic sulfate. Crit Rev Clin Lab Sci. 2000;37:299–344.[CrossRef][Medline]

18. Holmes JH, Miller ES, Hlad CJJ. Serum and urine sulfate changes in uremia. Trans Am Soc Artif Intern Organs. 1960;6:163–70.

19. Kirschbaum B. Effect of hemodialysis on the hypersulfatemia of chronic renal failure. ASAIO J. 1998;44:314–8.[Medline]

20. NRC. Nutrient requirements of poultry. 9th ed. Washington, DC: National Academies Press; 1994.

21. Constable PD. Clinical assessment of acid-base status: comparison of the Henderson-Hasselbalch and strong ion approaches. Vet Clin Pathol. 2000;29:115–28.[Medline]

22. Jandik P, Cheng J, Evrovski J, Avdalovic N. Simultaneous analysis of homocysteine and methionine in plasma. J Chromatogr B Biomed Sci Appl. 2001;759:145–51.[Medline]

23. Titgemeyer EC, Merchen NR, Berger LL. Estimation of lysine and methionine requirements of growing steers fed corn silage-based or corn-based diets. J Dairy Sci. 1988;71:421–34.[Abstract/Free Full Text]

24. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–54.[CrossRef][Medline]

25. Tietze F. Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal Biochem. 1969;27:502–22.[CrossRef][Medline]

26. Finkel KW, DuBose TD Jr. Metabolic acidosis. In: Dubose TD Jr, Hamm LL, editors. Acid-base and electrolyte disorders: a companion to Brenner and Rector's the kidney. Philadelphia: Saunders; 2002. p. 55–66.

27. Schwartz SM, Carroll HM, Scharschmidt LA. Sublimed (inorganic) sulfur ingestion. A cause of life-threatening metabolic acidosis with a high anion gap. Arch Intern Med. 1986;146:1437–8.[Abstract/Free Full Text]

28. Dudas PL, Renfro JL. Transepithelial sulfate transport by avian renal proximal tubule epithelium in primary culture. Am J Physiol Regul Integr Comp Physiol. 2002;283:R1354–61.[Abstract/Free Full Text]

29. Pelis RM, Renfro JL. Role of tubular secretion and carbonic anhydrase in vertebrate renal sulfate excretion. Am J Physiol Regul Integr Comp Physiol. 2004;287:R491–501.[Abstract/Free Full Text]

30. Stipanuk MH, Dominy JE Jr, Lee JI, Coloso RM. Mammalian cysteine metabolism: new insights into regulation of cysteine metabolism. J Nutr. 2006;136:S1652–9.[Abstract/Free Full Text]

31. Ercal N, Treeratphan P, Hammond TC, Matthews RH, Grannemann NH, Spitz DR. In vivo indices of oxidative stress in lead-exposed C57BL/6 mice are reduced by treatment with meso-2,3-dimercaptosuccinic acid or N-acetylcysteine. Free Radic Biol Med. 1996;21:157–61.[CrossRef][Medline]

32. Dilger RN, Baker DH. Oral N-acetyl-L-cysteine is a safe and effective precursor of cysteine. J Anim Sci. 2007;85:1712–8.[Abstract/Free Full Text]





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